Modified Fc molecules

ABSTRACT

Disclosed is a process for preparing a pharmacologically active compound, in which at least one internal conjugation site of an Fc domain sequence is selected that is amenable to conjugation of an additional functional moiety by a defined conjugation chemistry through the side chain of an amino acid residue at the conjugation site. An appropriate amino acid residue for conjugation may be present in a native Fc domain at the conjugation site or may be added by insertion (i.e., between amino acids in the native Fc domain) or by replacement (i.e., removing amino acids and substituting different amino acids). In the latter case, the number of amino acids added need not correspond to the number of amino acids removed from the previously existing Fc domain. This technology may be used to produce useful compositions of matter and pharmaceutical compositions containing them. A DNA encoding the inventive composition of matter, an expression vector containing the DNA, and a host cell containing the expression vector are also disclosed.

This application is a continuation of U.S. Ser. No. 13/171,233, filedJun. 28, 2011, now pending, which is a divisional of U.S. Ser. No.11/502,761, filed Aug. 10, 2006, now U.S. Pat. No. 8,008,453, whichclaims the benefit of U.S. Provisional Application No. 60/707,842, filedAug. 12, 2005, all of which are hereby incorporated by reference.

This application incorporates by reference all subject matter containedin this text file, which is identified by the name of the file,A-1037-US-CNT_SeqList_ST25.txt created on Jul. 9, 2015, the size ofwhich file is 230 KB.

Throughout this application various publications are referenced withinparentheses or brackets. The disclosures of these publications in theirentireties are hereby incorporated by reference in this application inorder to more fully describe the state of the art to which thisinvention pertains.

BACKGROUND OF THE INVENTION

1. Field of Art

The present invention relates to the biochemical arts, particularly toconjugates with immunoglobulin Fc domains.

2. Discussion of Related Art

The immunoglobulin Fc domain has found widespread use as a carrierprotein for a variety of therapeutic and diagnostic molecules.Antibodies comprise two functionally independent parts, a variabledomain known as “Fab”, which binds antigen, and a constant domain knownas “Fc”, which links to such effector functions as complement activationand attack by phagocytic cells. An Fc has a long serum half-life,whereas a Fab is short-lived. (Capon et al. (1989), Nature 337: 525-31).When constructed together with a therapeutic protein or peptide, an Fcdomain can provide longer half-life, or can incorporate such functionsas Fc receptor binding, protein A binding, complement fixation andperhaps even placental transfer. Id.

Numerous fusions of proteins and peptides have been engineered at eitherthe amino- or carboxy-termini. Also, a variety of enzymes and syntheticreporter molecules have been chemically conjugated to the side chains ofnon-terminal amino acids as well as the derivatized carbohydratemoieties of the Fc domain. Further, several polymers, such aspolyethylene glycol (PEG) have been conjugated to the Fc domain for thepurpose of improved half-life in vivo and reduced immunogenicity.

The success of the drug Enbrel® (etanercept) brought to fruition thepromise of therapeutic agents modified with the constant domain of anantibody. Table 1 summarizes several examples of the use of Fc fusionproteins known in the art.

TABLE 1 Fc fusion with therapeutic proteins Therapeutic Form of FcFusion partner implications Reference IgG1 N-terminus of Hodgkin'sdisease; U.S. Pat. No. CD30-L anaplastic lymphoma; 5,480,981 T-cellleukemia Murine Fcγ2a IL-10 Anti-inflammatory; Zheng et al., (1995), J.transplant rejection Immunol.154: 5590-600 IgG1 TNF receptor Septicshock Fisher et al. (1996), N. Engl. J. Med. 334: 1697-1702; Van Zee, K.et al. (1996), J. Immunol. 156: 2221-30 IgG, IgA, TNF receptorInflammation, U.S. Pat. No. IgM, or IgE autoimmune disorders 5,808,029,issued (excluding Sep. 15, 1998 the first domain) IgG1 CD4 receptor AIDSCapon et al. (1989), Nature 337: 525-31 IgG1, IgG3 N-terminus ofAnti-cancer, antiviral Harvill et al. (1995), IL-2 Immunotech. 1: 95-105IgG1 C-terminus of osteoarthritis; bone WO 97/23614, OPG densitypublished Jul. 3, 1997 IgG1 N-terminus of Anti-obesity WO 98/28427,filed leptin Dec. 11, 1997 Human Ig CTLA-4 Autoimmune disorders Linsley(1991), J. Exp. C 1 Med. 174: 561-9

A more recent development is fusion of randomly generated peptides withthe Fc domain. See U.S. Pat. No. 6,660,843, issued Dec. 9, 2003 to Feigeet al. (incorporated by reference in its entirety). Such molecules havecome to be known as “peptibodies.” They include one or more peptideslinked to the N-terminus, C-terminus, amino acid side chains, or to morethan one of these sites. Peptibody technology enables design oftherapeutic agents that incorporate peptides that target one or moreligands or receptors, tumor-homing peptides, membrane-transportingpeptides, and the like. Peptibody technology has proven useful in designof a number of such molecules, including linear anddisulfide-constrained peptides, “tandem peptide multimers” (i.e., morethan one peptide on a single chain of an Fc domain). See, for example,U.S. Pat. No. 6,660,843; U.S. Pat. App. No. 2003/0195156 A1, publishedOct. 16, 2003 (corresponding to WO 02/092620, published Nov. 21, 2002);U.S. Pat. App. No. 2003/0176352, published Sep. 18, 2003 (correspondingto WO 03/031589, published Apr. 17, 2003); U.S. Ser. No. 09/422,838,filed Oct. 22, 1999 (corresponding to WO 00/24770, published May 4,2000); U.S. Pat. App. No. 2003/0229023, published Dec. 11, 2003; WO03/057134, published Jul. 17, 2003; U.S. Pat. App. No. 2003/0236193,published Dec. 25, 2003 (corresponding to PCT/US04/010989, filed Apr. 8,2004); U.S. Ser. No. 10/666,480, filed Sep. 18, 2003 (corresponding toWO 04/026329, published Apr. 1, 2004), U.S. Patent App. No.2006/0140934, published Jun. 29, 2006 (corresponding to WO 2006/036834,published Apr. 4, 2006), each of which is hereby incorporated byreference in its entirety. The art would benefit from further technologyenabling such rational design of polypeptide therapeutic agents.

Conventional approaches for chemical conjugation to the immunoglobulinFc domain include random coupling to naturally occurring primary aminessuch as lysine and the amino-terminus or carboxylic acids such asglutamic acid, aspartic acid and the carboxy terminus. Alternatively,semi-selective site-specific coupling may be achieved through N-terminalconjugation under appropriate conditions, or derivatized carbohydratesas found on Fc proteins isolated from eukaryotic sources, or by partialreduction and coupling of native cysteine residues. (E.g., Kim et al., Apharmaceutical composition comprising an immunoglobulin Fc region as acarrier, WO 2005/047337). While each of these approaches has beenapplied successfully, they typically suffer from varying degrees ofconjugate heterogeneity, relatively low yields and sometimes,significant losses in functional activity are also observed. The artwould benefit from a process for selective, site-specific conjugation tothe immunoglobulin Fc domain without significant loss in functionalactivity.

SUMMARY OF THE INVENTION

The present invention concerns compositions of matter and a process formaking them. The inventive composition of matter, which is apharmacologically active compound, comprises a monomeric or multimericFc domain having at least one additional functional moiety that iscovalently bound (or conjugated), either directly or through a linker,to one or more specifically selected conjugation site(s) in the Fcdomain through the side chain of an amino acid residue at theconjugation site(s). Such an internal conjugation site may be alreadypresent in a native Fc domain sequence or can be added by insertion(i.e., between amino acids in the native Fc domain) or by replacement(i.e., removing amino acid residue(s) and substituting differentcanonical and/or non-canonical amino acid residue(s)) in the native Fcdomain sequence in order to create or “engineer” the conjugation site.In the latter case, the number of amino acid residues added need notcorrespond to the number of amino acid residues removed from thepreviously existing Fc domain sequence.

This inventive process of preparing a pharmacologically active compoundcomprising an Fc domain includes:

a. selecting at least one internal conjugation site of an Fc domainsequence, said conjugation site being amenable to conjugation of anadditional moiety by a defined coupling chemistry through the side chainof an amino acid residue at the conjugation site; and

b. conjugating a predetermined functional moiety to the selectedconjugation site by employing the defined conjugation chemistry.

In some embodiments, the functional moiety is a half-life extendingmoiety and/or a pharmacologically active moiety, which can be, forexample, a polypeptide, a peptide, a peptidomimetic, or a non-peptideorganic moiety. In other embodiments the additional functional moiety isa moiety detectably labeled with a radioisotope, an enzyme (e.g., aperoxidase or a kinase), a biotinyl moiety, a fluorophore, or achromophore. Alternatively, the additional functional moiety is animmobilized substrate, such as but not limited to, a plate surface, abead, a particle, a microparticle, a nanoparticle, a chip, a liposome, amatrix, or the like, provided that in a chain of additional functionalmoieties, the immobilized substrate is the additional moiety most distalfrom the Fc domain, and there can be no more than one immobilizedsubstrate in the chain.

The inventive process can be employed to modify an Fc domain that isalready linked through an N- or C-terminus or side chain to apolypeptide (e.g., a soluble fragment of TNF-R2, as in etanercept) or toa peptide (e.g., as described in U.S. Pat. App. Nos. 2003/0195156 A1,2003/0176352, 2003/0229023, and 2003/0236193; WO 00/24770; WO04/026329). The process described throughout can also be employed tomodify an Fc domain that is part of an antibody (e.g., adalimumab,epratuzumab, infliximab, Herceptin®, and the like). In this way,different molecules can be produced that have additionalfunctionalities, such as a binding domain to a different epitope, anadditional binding domain to the precursor molecule's existing epitope,or an additional half-life extending moiety.

The compounds of this invention may be prepared by standard syntheticmethods, recombinant DNA techniques, or any other methods of preparingpeptides and fusion proteins with reference to the disclosure of thisspecification.

The compounds of this invention may be used for therapeutic orprophylactic purposes by formulating them by methods known for otherproteinacious molecules and administering an effective amount to apatient, such as a human (or other mammal) in need thereof. Otherrelated aspects are also included in the current invention.

Numerous additional aspects and advantages of the present invention willbecome apparent upon consideration of the figures and detaileddescription of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a preferred subset (highlighted) of amino acid residuepositions for modification as conjugation site(s) from the superset ofsolvent-exposed surface residues (see Table 2) in a human IgG1 Fc domainsequence (SEQ ID NO:600). Underlined residues are not present in crystalstructure 1FC1, and many of these residues are good candidates formodification (e.g., insertion or substitution of amino acid residues)for creation of a conjugation site in accordance with the presentinvention, particularly the following amino acid residues from theaminoterminal fragment (first eighteen amino acid residues): DKTHTC . .. C . . . A . . . E . . . GG, i.e., through a side chain in thesubsequence at positions 1 through 6 of SEQ ID NO:600, at position 9 ofSEQ ID NO:600, at position 11 of SEQ ID NO:600, at position 13 of SEQ IDNO:600, at position 16 of SEQ ID NO:600, at position 17 of SEQ IDNO:600, and the following amino acids from the carboxy terminalfragment: GK, i.e., a non-terminal site at position 226 of SEQ IDNO:600, or position 227 of SEQ ID NO:600.

FIG. 2 shows (SEQ ID NO:599), the sequence of human IgG1 Fc monomer withpredicted loop region sequences in boldface; the N-terminal methionineis added for expression from E. coli and is not otherwise present in thenative IgG1 sequence (reference sequence SEQ ID NO:600). Amino acidresidues useful as preferred sites for insertion or substitution ofamino acid residues for creation of a conjugation site in accordancewith the present invention are underlined.

FIG. 3 shows (SEQ ID NO:599), the sequence of human IgG1 Fc monomer withpredicted loop region sequences in boldface. Amino acid residuepositions useful as conjugation sites in accordance with the presentinvention also include the underlined. Preferred surface exposedconjugation sites selected from FIG. 1 are indicated by highlightinghere.

FIG. 4 shows proposed cysteine mutation sites mapped to Fc structure.The amino acid residues identified by arrows are designated positionsrelative to reference sequence SEQ ID NO:599, as follows:

A: Ser 196, which is the most solvent-exposed and is in a rigid helix.

B: Gln 143, which is a deep polar pocket and is from the same strand asCys 148.

C: Leu 139, which is an Fc-loop region, and is near the C-terminus in apolar pocket.

D: Ser 145, which is from the same strand as cys 148 and is in a polarpocket on a β-sheet surface in a cleft between subunits.

FIG. 5 shows SDS-PAGE gel analysis (4-20% Tris:Glycine polyacrylamidegel for 1.5 hours at 125V, 35 mA, 0.1% SDS) of purified huFc-cysteineanalogs described in Example 2. Lanes: 1, 8 contained MW markers, lanes2, 9 contained clone 13300 Fc(Q143C), lanes 3, 10 contained clone 13322.Fc(L139C), lanes 4, 11 contained clone 13323 Fc(S145C) and lanes 5, 12contained clone 13324 Fc(S196C). Lanes 2-6 were reduced and lanes 9-12were non-reduced.

FIG. 6 shows purity by SEC-HPLC analyses of clone 13324 huFc(S196C) asdescribed in Example 2. Samples (20 ng) were eluted in 100 mM sodiumphosphate, 150 mM NaCl, pH 6.9 on a TSK G3000SW×1 column, 7.8 mm ID×30cm, 5 μm bead size) at 0.5 ml/min.

FIG. 7 shows purity and mass determination by LC-MS of clone 13324huFc(S196C) as described in Example 2. Samples (20 ng) were eluted in0.1% TFA with a linear 0-90% acetonitrile gradient from a Zorbax300SB-C18 column, 2.1 mm×150 cm.

FIG. 8A shows non-reduced SDS-PAGE analysis of huFc (S196C) analogPEGylated after varying degrees of TCEP reduction, as described inExample 3. Molar stoichiometries of engineered Cysteine: TCEP were: 1:0in lane 1, 1:0.5 in lane 3, 1:0.75 in lane 4, 1:1 in lane 5, 1:1.25 inlane 6, 1:1.5 in lane 7, 1:2 in lane 8 and 1:5 in lane 9. MW markerswere in lane 2. 2 μg of non-reduced protein were loaded to each lane andrun in 4-20% Tris-Glycine polyacrylamide gel with 0.1% SDS at 125 V, 35mA and 5 W, for 1.5 hours.

FIG. 8B shows reduced SDS-PAGE analysis of huFc (S196C) analog PEGylatedafter varying degrees of TCEP reduction, as described in Example 3.Molar stoichiometries of engineered Cysteine: TCEP were: 1:0 in lane 2,1:0.5 in lane 3, 1:0.75 in lane 4, 1:1 in lane 5, 1:1.25 in lane 6,1:1.5 in lane 7, 1:2 in lane 8 and 1:5 in lane 9. MW markers were inlane 1. 2 μg of reduced protein were loaded to each lane and run in4-20% Tris-Glycine polyacrylamide gel with 0.1% SDS at 125 V, 35 mA and5 W, for 1.5 hours.

FIG. 9A-B shows SEC-HPLC analyses of of huFc (S196C) analog PEGylatedafter varying degrees of TCEP reduction, as described in Example 3. 20μg protein were loaded to TSK 3000SW×1 column (7.8 mm×30 cm, 5 micron)and eluted in 100 mM sodium phosphate, 150 mM NaCl, pH 6.9.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS OF THE INVENTIONDefinition of Terms

The terms used throughout this specification are defined as follows,unless otherwise limited in specific instances. As used in thespecification and the appended claims, the singular forms “a”, “an”, and“the” include plural referents unless the context clearly dictatesotherwise. Unless specified otherwise, the left-hand end ofsingle-stranded polynucleotide sequences is the 5′ end; the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction; sequenceregions on the DNA strand having the same sequence as the RNA and whichare 5′ to the 5′ end of the RNA transcript are referred to as “upstreamsequences”; sequence regions on the DNA strand having the same sequenceas the RNA and which are 3′ to the 3′ end of the RNA transcript arereferred to as “downstream sequences”.

When used in connection with an amino acid sequence, the term“comprising” means that a compound may include additional amino acidresidues on either or both of the N- or C-termini of the given sequence.

A conjugation site being “amenable to conjugation” means that the sidechain of the amino acid residue at the selected conjugation site willreact with the additional functional moiety of interest (or with alinker covalently attached to the additional functional moiety), underthe defined chemical conditions, resulting in covalent binding of theadditional functional moiety (directly or via the linker) to the sidechain as a major reaction product.

“Antibody” or “antibody peptide(s)” refer to an intact antibody, or abinding fragment thereof that competes with the intact antibody forspecific binding and includes chimeric, humanized, fully human, andbispecific antibodies. In certain embodiments, binding fragments areproduced by recombinant DNA techniques. In additional embodiments,binding fragments are produced by enzymatic or chemical cleavage ofintact antibodies. Binding fragments include, but are not limited to,Fab, Fab′, F(ab′)₂, Fv, and single-chain antibodies.

The inventive composition comprises a Fc domain having at least oneadditional functional moiety covalently bound to the Fc domain. The term“Fe domain” encompasses native Fc and Fc variant molecules and sequencesas defined herein below. As with Fc variants and native Fe's, the term“Fe domain” includes molecules in monomeric or multimeric form, whetherdigested from whole antibody or produced by other means. In oneembodiment, the Fc domain is a human native Fc domain. In otherembodiments, the “Fe domain” can be a Fc variant, an analog, a mutant, atruncation, or a derivative of human Fc or of an alternative mammalianFc polypeptide.

The term “native Fc” refers to a molecule or sequence comprising theamino acid sequence of a non-antigen-binding fragment resulting fromdigestion of whole antibody, whether in monomeric or multimeric form, atwhich a peptide may be added or conjugated by being covalently bound,directly or indirectly through a linker, to a loop region of the Fcdomain. The original immunoglobulin source of the native Fc ispreferably of human origin (although non-human mammalian native Fc isincluded in “native Fc” and can also be useful in some embodiments), andmay be any of the immunoglobulins, although IgG1 and IgG2 are preferred.The native Fc may optionally comprise an amino terminal methionineresidue. By way of example, SEQ ID NO:600 is the native human IgG1sequence (used, in some cases, as a reference sequence herein), and Fcvariant SEQ ID NO:599 (also used in some cases as a reference sequenceherein) is the same sequence with an amino terminal methionine residue.Native Fcs are made up of monomeric polypeptides that may be linked intodimeric or multimeric forms by covalent (i.e., disulfide bonds) andnon-covalent association. The number of intermolecular disulfide bondsbetween monomeric subunits of native Fc molecules ranges from 1 to 4depending on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2,IgG3, IgA1, IgGA2). One example of a native Fc is a disulfide-bondeddimer resulting from papain digestion of an IgG (see Ellison et al.(1982), Nucleic Acids Res. 10: 4071-9). The term “native Fc” as usedherein is generic to the monomeric, dimeric, and multimeric forms.

Statements or claims concerning amino acid residue positions citedherein relative to one particular “reference sequence” (i.e., SEQ IDNO:600 or SEQ ID NO:599) apply equally to the corresponding position inthe other reference sequence, or in a different native Fc sequence, Fcvariant sequence, or other modified Fc domain sequence, in an alignmentof the two (i.e., comparing the recited reference sequence and thesecond Fc domain sequence of interest), e.g., position 2 in SEQ IDNO:599 corresponds to position 1 in SEQ ID NO:600, etc.

The term “Fc variant” refers to a molecule or sequence that is modifiedfrom a native Fc but still comprises a binding site for the salvagereceptor, FcRn. International applications WO 97/34631 (published 25Sep. 1997) and WO 96/32478 describe exemplary Fc variants, as well asinteraction with the salvage receptor, and are hereby incorporated byreference. Thus, the term “Fc variant” comprises a molecule or sequencethat is humanized from a non-human native Fc. Furthermore, a native Fccomprises sites that may be removed because they provide structuralfeatures or biological activity that are not required for molecules ofthe present invention. Thus, the term “Fc variant” comprises a moleculeor sequence that lacks one or more native Fc sites or residues thataffect or are involved in (1) disulfide bond formation, (2)incompatibility with a selected host cell (3) N-terminal heterogeneityupon expression in a selected host cell, (4) glycosylation, (5)interaction with complement, (6) binding to an Fc receptor other than asalvage receptor, or (7) antibody-dependent cellular cytotoxicity(ADCC). Fc variants are described in further detail hereinafter.

The term “internal” conjugation site means that conjugation of the atleast one additional moiety, or moieties, is non-terminal, i.e., notthrough the α-amino site or the α-carboxy site of the Fc domain,although there optionally can also be additional moieties conjugatedterminally at the N-terminal and/or C-terminal of the Fc domain.

The term “loop” region or “Fc-loop” region refers to a primary sequenceof amino acid residues which connects two regions comprising secondarystructure, such as an α-helix or β-sheet, in the immediate N-terminaland C-terminal directions of primary structure from the loop region.Examples include, but are not limited to, CH2 or CH3 loop regions. Oneof skill in the art understands that a loop region, while not itselfcomprising secondary structure, may influence or contribute to secondaryor higher order protein structure.

The term “multimer” as applied to Fc domains or molecules comprising Fcdomains refers to molecules having two or more Fc domain polypeptidechains associated covalently, noncovalently, or by both covalent andnon-covalent interactions. IgG molecules typically form dimers; IgM,pentamers; IgD, dimers; and IgA, monomers, dimers, trimers, ortetramers. Multimers may be formed by exploiting the sequence andresulting activity of the native Ig source of the Fc or by derivatizing(as defined below) such a native Fc.

The term “dimer” as applied to Fc domains or molecules comprising Fcdomains refers to molecules having two polypeptide chains associatedcovalently or non-covalently. Exemplary dimers within the scope of thisinvention are as shown in U.S. Pat. No. 6,660,843, FIG. 2, which ishereby incorporated by reference. “Dimers” include homodimers andheterodimers.

The terms “derivatizing” and “derivative” or “derivatized” compriseprocesses and resulting compounds respectively in which (1) the compoundhas a cyclic portion; for example, cross-linking between cysteinylresidues within the compound; (2) the compound is cross-linked or has across-linking site; for example, the compound has a cysteinyl residueand thus forms cross-linked dimers in culture or in vivo; (3) one ormore peptidyl linkage is replaced by a non-peptidyl linkage; (4) theN-terminus is replaced by —NRR¹, NRC(O)R¹, —NRC(O)OR¹, —NRS(O)₂R¹,—NHC(O)NHR, a succinimide group, or substituted or unsubstitutedbenzyloxycarbonyl-NH—, wherein R and R¹ and the ring substituents are asdefined hereinafter; (5) the C-terminus is replaced by —C(O)R² or —NR³R⁴wherein R², R³ and R⁴ are as defined hereinafter; and (6) compounds inwhich individual amino acid moieties are modified through treatment withagents capable of reacting with selected side chains or terminalresidues. Derivatives are further described hereinafter.

The term “half-life extending moiety refers to a pharmaceuticallyacceptable moiety, domain, or “vehicle” covalently linked or conjugatedto the Fc domain and/or a pharmaceutically active moiety, that preventsor mitigates in vivo proteolytic degradation or otheractivity-diminishing chemical modification of the pharmaceuticallyactive moiety, increases half-life or other pharmacokinetic propertiessuch as but not limited to increasing the rate of absorption, reducestoxicity, improves solubility, increases biological activity and/ortarget selectivity of the pharmaceutically active moiety with respect toa target of interest, increases manufacturability, and/or reducesimmunogenicity of the pharmaceutically active moiety (e.g., a peptide ornon-peptide moiety), compared to an unconjugated form of thepharamaceutically active moiety. Polyethylene glycol (PEG) is an exampleof a useful half-life extending moiety. Other examples of the half-lifeextending moiety, in accordance with the invention, include a copolymerof ethylene glycol, a copolymer of propylene glycol, acarboxymethylcellulose, a polyvinyl pyrrolidone, a poly-1,3-dioxolane, apoly-1,3,6-trioxane, an ethylene maleic anhydride copolymer, apolyaminoacid (e.g., polylysine), a dextran n-vinyl pyrrolidone, a polyn-vinyl pyrrolidone, a propylene glycol homopolymer, a propylene oxidepolymer, an ethylene oxide polymer, a polyoxyethylated polyol, apolyvinyl alcohol, a linear or branched glycosylated chain, apolyacetal, a long chain fatty acid, a long chain hydrophobic aliphaticgroup, an immunoglobulin F_(c) domain (see, e.g., Feige et al., Modifiedpeptides as therapeutic agents, U.S. Pat. No. 6,660,843), an albumin(e.g., human serum albumin; see, e.g., Rosen et al., Albumin fusionproteins, U.S. Pat. No. 6,926,898 and US 2005/0054051; Bridon et al.,Protection of endogenous therapeutic peptides from peptidase activitythrough conjugation to blood components, U.S. Pat. No. 6,887,470), atransthyretin (TTR; see, e.g., Walker et al., Use of transthyretinpeptide/protein fusions to increase the serum half-life ofpharmacologically active peptides/proteins, US 2003/0195154 A1;2003/0191056 A1), or a thyroxine-binding globulin (TBG).

Other embodiments of the useful half-life extending moiety, inaccordance with the invention, include peptide ligands or small(non-peptide organic) molecule ligands that have binding affinity for along half-life serum protein under physiological conditions oftemperature, pH, and ionic strength. Examples include an albumin-bindingpeptide or small molecule ligand, a transthyretin-binding peptide orsmall molecule ligand, a thyroxine-binding globulin-binding peptide orsmall molecule ligand, an antibody-binding peptide or small moleculeligand, or another peptide or small molecule that has an affinity for along half-life serum protein. (See, e.g., Blaney et al., Method andcompositions for increasing the serum half-life of pharmacologicallyactive agents by binding to transthyretin-selective ligands, U.S. Pat.No. 5,714,142; Sato et al., Serum albumin binding moieties, US2003/0069395 A1; Jones et al., Pharmaceutical active conjugates, U.S.Pat. No. 6,342,225). A “long half-life serum protein” is one of thehundreds of different proteins dissolved in mammalian blood plasma,including so-called “carrier proteins” (such as albumin, transferrin andhaptoglobin), fibrinogen and other blood coagulation factors, complementcomponents, immunoglobulins, enzyme inhibitors, precursors of substancessuch as angiotensin and bradykinin and many other types of proteins. Theinvention encompasses the use of any single species of pharmaceuticallyacceptable half-life extending moiety, such as, but not limited to,those described herein, or the use of a combination of two or moredifferent half-life extending moieties.

The term “polypeptide” refers to molecules of greater than 40 aminoacids, whether existing in nature or not, provided that such moleculesare not membrane-bound. Exemplary polypeptides include interleukin(IL)-1ra, leptin, soluble tumor necrosis factor (TNF) receptors type 1and type 2 (sTNF-R1, sTNF-R2), keratinocyte growth factor (KGF),erythropoietin (EPO), thrombopoietin (TPO), granulocytecolony-stimulating factor (G-CSF), darbepoietin, glial cell line-derivedneurotrophic factor (GDNF), Fab fragments and the like. “Polypeptide”and “protein” are used interchangeably herein.

The term “peptide” refers to molecules of 2 to 40 amino acid residues inlength, with molecules of 3 to 40 amino acid residues or 6 to 40 aminoacid residues in length preferred. Exemplary peptides may be randomlygenerated by any of the methods cited above, carried in a peptidelibrary (e.g., a phage display library), or derived by digestion ofproteins. “Peptides” include cyclic peptides.

In further describing peptides or polypeptides herein, a one-letterabbreviation system is frequently applied to designate the identities ofthe twenty “canonical” amino acid residues generally incorporated intonaturally occurring peptides and proteins (Table 1A). Such one-letterabbreviations are entirely interchangeable in meaning with three-letterabbreviations, or non-abbreviated amino acid names. Within theone-letter abbreviation system used herein, an uppercase letterindicates a L-amino acid, and a lower case letter indicates a D-aminoacid, unless otherwise noted herein. For example, the abbreviation “R”designates L-arginine and the abbreviation “r” designates D-arginine.

TABLE 1A One-letter abbreviations for the canonical amino acids.Three-letter abbreviations are in parentheses. Alanine (Ala) A Glutamine(Gln) Q Leucine (Leu) L Serine (Ser) S Arginine (Arg) R Glutamic Acid(Glu) E Lysine (Lys) K Threonine (Thr) T Asparagine (Asn) N Glycine(Gly) G Methionine (Met) M Tryptophan (Trp) W Aspartic Acid (Asp) DHistidine (His) H Phenylalanine (Phe) F Tyrosine (Tyr) Y Cysteine (Cys)C Isoleucine (Ile) I Proline (Pro) P Valine (Val) V

An amino acid substitution in an amino acid sequence is typicallydesignated herein with a one-letter abbreviation for the amino acidresidue in a particular position, followed by the numerical amino acidposition relative to the native peptide or polypeptide sequence ofinterest, which is then followed by the one-letter symbol for the aminoacid residue substituted in. For example, “T30D” symbolizes asubstitution of a threonine residue by an aspartate residue at aminoacid position 30, relative to a hypothetical native peptide orpolypeptide sequence. By way of further example, “R18hR” or “R18Cit”indicates a substitution of an arginine residue by a homoarginine or acitrulline residue, respectively, at amino acid position 18, relative tothe hypothetical native peptide or polypeptide. An amino acid positionwithin the amino acid sequence of any particular poly peptide or peptide(or peptide analog) described herein may differ from its positionrelative to the native sequence, i.e., as determined in an alignment ofthe N-terminal or C-terminal end of the peptide's amino acid sequencewith the N-terminal or C-terminal end, as appropriate, of the nativepolypeptide or peptide sequence.

The term “non-canonical amino acid residue” refers to amino acidresidues in D- or L-form that are not among the 20 canonical amino acidsgenerally incorporated into naturally occurring proteins. Non-canonicalamino acids include naturally rare (in peptides or proteins) amino acidresidues or unnatural amino acid residues. Example of non-canonicalamino acids include, without limitation, β-amino acids, homoamino acids,cyclic amino acids, α-, α-disubstituted amino acids, N-alkyl aminoacids, and amino acids with derivatized side chains. Other examplesinclude (in the L-form or D-form): citrulline (Cit), homocitrulline(hCit), N-methylcitrulline (NMeCit), N-methylhomocitrulline (NMeHoCit),ornithine (Orn or O), N-Methylornithine (NMeOrn), sarcosine (Sar),homolysine (hK or Hlys), homoarginine (hR or hArg), homoglutamine (hQ),N-methylarginine (NMeR), N-methylleucine (NMeL), N-methylhomolysine(NMeHoK), N-methylglutamine (NMeQ), norleucine (Nle), norvaline (Nva),1,2,3,4-tetrahydroisoquinoline (Tic), nitrophenylalanine (nitrophe),aminophenylalanine (aminophe), benzylphenyalanine (benzylphe),γ-carboxyglutamic acid (γ-carboxyglu), hydroxyproline (hydroxypro),p-carboxyl-phenylalanine (Cpa), α-aminoadipic acid (Aad), acetylarginine(acetylarg), α, β-diaminopropionoic acid (Dpr), α, γ-diaminobutyric acid(Dab), diaminopropionic acid (Dap), β-(1-Naphthyl)-alanine (1Na1),β-(2-Naphthyl)-alanine (2Na1), cyclohexylalanine (Cha),4-methyl-phenylalanine (MePhe), 3,3-diphenyl-alanine (BiPhA),aminobutyric acid (Abu), 4-phenyl-phenylalanine (4Bip),α-amino-isobutyric acid (Aib), beta-alanine, beta-aminopropionic acid,piperidinic acid, aminocaprioic acid, aminoheptanoic acid, aminopimelicacid, desmosine, diaminopimelic acid, N-ethylglycine, N-ethylaspargine,hyroxylysine, allo-hydroxylysine, isodesmosine, allo-isoleucine,N-methylglycine, N-methylisoleucine, N-methylvaline, 4-hydroxyproline,γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine,O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine,5-hydroxylysine, ω-methylarginine, and other similar amino acids, andderivatized forms of any of these as described herein.

Nomenclature and Symbolism for Amino Acids and Peptides by the UPAC-IUBJoint Commission on Biochemical Nomenclature (JCBN) have been publishedin the following documents: Biochem. J., 1984, 219, 345-373; Eur. J.Biochem., 1984, 138, 9-37; 1985, 152, 1; 1993, 213, 2; Internat. J.Pept. Prot. Res., 1984, 24, following p 84; J. Biol. Chem., 1985, 260,14-42; Pure Appl. Chem., 1984, 56, 595-624; Amino Acids and Peptides,1985, 16, 387-410; Biochemical Nomenclature and Related Documents, 2ndedition, Portland Press, 1992, pages 39-69. The term “protease” issynonymous with “peptidase”. Proteases comprise two groups of enzymes:the endopeptidases which cleave peptide bonds at points within theprotein, and the exopeptidases, which remove one or more amino acidsfrom either N- or C-terminus respectively. The term “proteinase” is alsoused as a synonym for endopeptidase. The four mechanistic classes ofproteinases are: serine proteinases, cysteine proteinases, asparticproteinases, and metallo-proteinases. In addition to these fourmechanistic classes, there is a section of the enzyme nomenclature whichis allocated for proteases of unidentified catalytic mechanism. Thisindicates that the catalytic mechanism has not been identified.

Cleavage subsite nomenclature is commonly adopted from a scheme createdby Schechter and Berger (Schechter I. & Berger A., On the size of theactive site in proteases. I. Papain, Biochemical and BiophysicalResearch Communication, 27:157 (1967); Schechter I. & Berger A., On theactive site of proteases. 3. Mapping the active site of papain; specificinhibitor peptides of papain, Biochemical and Biophysical ResearchCommunication, 32:898 (1968)). According to this model, amino acidresidues in a substrate undergoing cleavage are designated P1, P2, P3,P4 etc. in the N-terminal direction from the cleaved bond. Likewise, theresidues in the C-terminal direction are designated P1′, P2′, P3′, P4′.etc.

The skilled artisan is aware of a variety of tools for identifyingprotease binding or protease cleavage sites of interest. For example,the PeptideCutter software tool is available through the ExPASy (ExpertProtein Analysis System) proteomics server of the Swiss Institute ofBioinformatics (SIB; expasy.org/tools/peptidecutter). PeptideCuttersearches a protein sequence from the SWISS-PROT and/or TrEMBL databasesor a user-entered protein sequence for protease cleavage sites. Singleproteases and chemicals, a selection or the whole list of proteases andchemicals can be used. Different forms of output of the results areavailable: tables of cleavage sites either grouped alphabeticallyaccording to enzyme names or sequentially according to the amino acidnumber. A third option for output is a map of cleavage sites. Thesequence and the cleavage sites mapped onto it are grouped in blocks,the size of which can be chosen by the user. Other tools are also knownfor determining protease cleavage sites. (E.g., Turk, B. et al.,Determination of protease cleavage site motifs using mixture-basedoriented peptide libraries, Nature Biotechnology, 19:661-667 (2001);Barrett A. et al., Handbook of proteolytic enzymes, Academic Press(1998)).

The serine proteinases include the chymotrypsin family, which includesmammalian protease enzymes such as chymotrypsin, trypsin or elastase orkallikrein. The serine proteinases exhibit different substratespecificities, which are related to amino acid substitutions in thevarious enzyme subsites interacting with the substrate residues. Someenzymes have an extended interaction site with the substrate whereasothers have a specificity restricted to the P1 substrate residue.

Trypsin preferentially cleaves at R or K in position P1. A statisticalstudy carried out by Keil (1992) described the negative influences ofresidues surrounding the Arg- and Lys-bonds (i.e. the positions P2 andP1′, respectively) during trypsin cleavage. (Keil, B., Specificity ofproteolysis, Springer-Verlag Berlin-Heidelberg-NewYork, 335 (1992)). Aproline residue in position P1′ normally exerts a strong negativeinfluence on trypsin cleavage. Similarly, the positioning of R and K inP1′ results in an inhibition, as well as negatively charged residues inpositions P2 and P1′.

Chymotrypsin preferentially cleaves at a W, Y or F in position P1 (highspecificity) and to a lesser extent at L, M or H residue in position P1.(Keil, 1992). Exceptions to these rules are the following: When W isfound in position P1, the cleavage is blocked when M or P are found inposition P1′ at the same time. Furthermore, a proline residue inposition P1′ nearly fully blocks the cleavage independent of the aminoacids found in position P1. When an M residue is found in position P1,the cleavage is blocked by the presence of a Y residue in position P1′.Finally, when H is located in position P1, the presence of a D, M or Wresidue also blocks the cleavage.

Membrane metallo-endopeptidase (NEP; neutral endopeptidase,kidney-brush-border neutral proteinase, enkephalinase, EC 3.4.24.11)cleaves peptides at the amino side of hydrophobic amino acid residues.(Connelly, J C et al., Neutral Endopeptidase 24.11 in Human Neutrophils:Cleavage of Chemotactic Peptide, PNAS, 82(24):8737-8741 (1985)).

Thrombin preferentially cleaves at an R residue in position P1. (Keil,1992). The natural substrate of thrombin is fibrinogen. Optimum cleavagesites are when an R residue is in position P1 and Gly is in position P2and position P1′. Likewise, when hydrophobic amino acid residues arefound in position P4 and position P3, a proline residue in position P2,an R residue in position P1, and non-acidic amino acid residues inposition P1′ and position P2′. A very important residue for its naturalsubstrate fibrinogen is a D residue in P10.

Caspases are a family of cysteine proteases bearing an active site witha conserved amino acid sequence and which cleave peptides specificallyfollowing D residues. (Earnshaw W C et al., Mammalian caspases:Structure, activation, substrates, and functions during apoptosis,Annual Review of Biochemistry, 68:383-424 (1999)).

The Arg-C proteinase preferentially cleaves at an R residue in positionP1. The cleavage behavior seems to be only moderately affected byresidues in position P1′. (Keil, 1992). The Asp-N endopeptidase cleavesspecifically bonds with a D residue in position P1′. (Keil, 1992).

The foregoing is merely exemplary and by no means an exhaustivetreatment of knowledge available to the skilled artisan concerningprotease binding and/or cleavage sites that the skilled artisan may beinterested in eliminating in practicing the invention.

The term “randomized” as used to refer to peptide sequences refers torandom sequences (e.g., selected by phage display methods) and sequencesin which one or more residues of a naturally occurring molecule isreplaced by an amino acid residue not appearing in that position in thenaturally occurring molecule. Exemplary methods for identifying peptidesequences include phage display, E. coli display, ribosome display,yeast-based screening, RNA-peptide screening, chemical screening,rational design, protein structural analysis, and the like.

A peptidomimetic can include a small peptide-like chain that containsone or more amide bond isosteres and can contain both natural andunnatural amino acids. Such peptide-like peptidomimetics typically arisefrom modification of an existing polypeptide or peptide in order toalter the molecule's properties. For example, they may arise frommodifications to change the molecule's stability or biological activity.These modifications involve changes to the peptide that will not occurnaturally (such as incorporation of unnatural amino acids).Alternatively, peptidomimetics include non-peptide small moleculeshaving peptide-like biochemical or pharmacological activity and/orchemical structure, such as, but not limited to, steric structure. Anexample of such a peptidomimetic compound is BIBN 4096 BS.

The term “pharmacologically active” means that a substance so describedis determined to have activity that affects a medical parameter (e.g.,blood pressure, blood cell count, cholesterol level) or disease state(e.g., cancer, autoimmune disorders, neurological disorders, chronicpain). Thus, pharmacologically active peptides or polypeptides compriseagonistic or mimetic and antagonistic peptides as defined below.

The terms “-mimetic peptide” and “-agonist peptide” refer, respectively,to a peptide or polypeptide having biological activity comparable to aprotein (e.g., EPO, TPO, G-CSF) of interest or to a peptide orpolypeptide that interacts as an agonist with a particular protein ofinterest. These terms further include peptides or polypeptides thatindirectly mimic the activity of a protein of interest, such as bypotentiating the effects of the natural ligand of the protein ofinterest; see, for example, the EPO-mimetic peptides listed in Table 5hereof and in U.S. Pat. No. 6,660,843, which is hereby incorporated byreference. Thus, the term “EPO-mimetic peptide” comprises any peptidesor polypeptides that can be identified or derived as described inWrighton et al. (1996), Science 273: 458-63, Naranda et al. (1999),Proc. Natl. Acad. Sci. USA 96: 7569-74, or any other reference in Table5 identified as having EPO-mimetic subject matter. Those of ordinaryskill in the art appreciate that each of these references enables one toselect different peptides or polypeptides than actually disclosedtherein by following the disclosed procedures with different peptidelibraries.

The term “-antagonist peptide” or “inhibitor peptide” refers to apeptide that blocks or in some way interferes with the biologicalactivity of the associated protein of interest, or has biologicalactivity comparable to a known antagonist or inhibitor of the associatedprotein of interest. Thus, the term “BAFF-antagonist peptide” comprisespeptides that can be identified or derived as described in U.S. Pat.Appln. No. 2003/0195156 A1, which is incorporated herein by referenceand those peptides appearing in Table 10. Those of ordinary skill in theart appreciate that the foregoing reference enables one to selectdifferent peptides than actually disclosed therein by following thedisclosed procedures with different peptide libraries.

In the inventive composition of composition matter, the monomeric ormultimeric Fc domain has at least one additional functional moiety thatis covalently bound (or conjugated) to one or more “specificallyselected” conjugation site(s) in the Fc domain. The term “specificallyselected” with respect to conjugation site means that the major productor derivative of the conjugation chemistry (or chemical reaction)employed is through the side chain of an amino acid residue at thespecifically selected conjugation site in the Fc domain. Minor reactionproducts can also result from the conjugation reaction, but these can bepurified out, if desired or appropriate. “Toxin peptides” includepeptides and polypeptides having the same amino acid sequence of anaturally occurring pharmacologically active peptide or polypeptide thatcan be isolated from a venom, and also include modified peptide analogsof such naturally occurring molecules. (See, e.g., Kalman et al.,ShK-DaP22, a potent Kv1.3-specific immunosuppressive polypeptide, J.Biol. Chem. 273(49):32697-707 (1998); Kem et al., U.S. Pat. No.6,077,680; Mouhat et al., OsK1 derivatives, WO 2006/002850 A2; Chandy etal., Analogs of SHK toxin and their uses in selective inhibition ofKv1.3 potassium channels, WO 2006/042151). Snakes, scorpions, spiders,bees, snails and sea anemone are a few examples of organisms thatproduce venom that can serve as a rich source of small bioactive toxinpeptides or “toxins” that potently and selectively target ion channelsand receptors.

The toxin peptides are usually between about 20 and about 80 amino acidsin length, contain 2-5 disulfide linkages and form a very compactstructure. Toxin peptides (e.g., from the venom of scorpions, seaanemones and cone snails) have been isolated and characterized for theirimpact on ion channels. Such peptides appear to have evolved from arelatively small number of structural frameworks that are particularlywell suited to addressing the critical issues of potency and stability.The majority of scorpion and Conus toxin peptides, for example, contain10-40 amino acids and up to five disulfide bonds, forming extremelycompact and constrained structure (microproteins) often resistant toproteolysis. The conotoxin and scorpion toxin peptides can be dividedinto a number of superfamilies based on their disulfide connections andpeptide folds. The solution structure of many of these has beendetermined by NMR spectroscopy, illustrating their compact structure andverifying conservation of their family fold. (E.g., Tudor et al.,Ionisation behaviour and solution properties of the potassium-channelblocker ShK toxin, Eur. J. Biochem. 251(1-2):133-41(1998); Pennington etal., Role of disulfide bonds in the structure and potassium channelblocking activity of ShK toxin, Biochem. 38(44): 14549-58 (1999);Jaravine et al., Three-dimensional structure of toxin OSK1 fromOrthochirus scrobiculosus scorpion venom, Biochem. 36(6):1223-32 (1997);del Rio-Portillo et al.; NMR solution structure of Cn12, a novel peptidefrom the Mexican scorpion Centruroides noxius with a typical beta-toxinsequence but with alpha-like physiological activity, Eur. J. Biochem.271(12): 2504-16 (2004); Prochnicka-Chalufour et al., Solution structureof discrepin, a new K+-channel blocking peptide from the alpha-KTx15subfamily, Biochem. 45(6):1795-1804 (2006)). Examples ofpharmacologically active toxin peptides for which the practice of thepresent invention can be useful include, but are not limited to ShK,OSK1, charybdotoxin (ChTx), kaliotoxinl KTX1), or maurotoxin, or toxinpeptide analogs of any of these, modified from the native sequences atone or more amino acid residues. Other examples are known in the art, orcan be found in U.S. patent application Ser. No. 11/406,454 (titled:Toxin Peptide Therapeutic Agents), filed on Apr. 17, 2006, which isincorporated by reference in its entirety.

The term “TPO-mimetic peptide” comprises peptides that can be identifiedor derived as described in Cwirla et al. (1997), Science 276: 1696-9,U.S. Pat. Nos. 5,869,451 and 5,932,946, which are incorporated byreference; U.S. Pat. App. No. 2003/0176352, published Sep. 18, 2003,which is incorporated by reference; WO 03/031589, published Apr. 17,2003; WO 00/24770, published May 4, 2000; and any peptides appearing inTable 6. Those of ordinary skill in the art appreciate that each ofthese references enables one to select different peptides than actuallydisclosed therein by following the disclosed procedures with differentpeptide libraries.

The term “ang-2-binding peptide” comprises peptides that can beidentified or derived as described in U.S. Pat. App. No. 2003/0229023,published Dec. 11, 2003; WO 03/057134, published Jul. 17, 2003; U.S.2003/0236193, published Dec. 25, 2003 (each of which is incorporatedherein by reference); and any peptides appearing in Table 7. Those ofordinary skill in the art appreciate that each of these referencesenables one to select different peptides than actually disclosed thereinby following the disclosed procedures with different peptide libraries.

The term “NGF-binding peptide” comprises peptides that can be identifiedor derived as described in WO 04/026329, published Apr. 1, 2004 and anypeptides identified in Table 8. Those of ordinary skill in the artappreciate that this reference enables one to select different peptidesthan actually disclosed therein by following the disclosed procedureswith different peptide libraries.

The term “myostatin-binding peptide” comprises peptides that can beidentified or derived as described in U.S. Ser. No. 10/742,379, filedDec. 19, 2003, which is incorporated herein by reference, and peptidesappearing in Table 9. Those of ordinary skill in the art appreciate thateach of these references enables one to select different peptides thanactually disclosed therein by following the disclosed procedures withdifferent peptide libraries.

“PEGylated peptide” is meant a peptide or protein having a polyethyleneglycol (PEG) moiety covalently bound to an amino acid residue of thepeptide itself or to a peptidyl or non-peptidyl linker (including butnot limited to aromatic linkers) that is covalently bound to a residueof the peptide.

By “polyethylene glycol” or “PEG” is meant a polyalkylene glycolcompound or a derivative thereof, with or without coupling agents orderivatization with coupling or activating moieties (e.g., withaldehyde, hydroxysuccinimidyl, hydrazide, thiol, triflate, tresylate,azirdine, oxirane, orthopyridyl disulphide, vinylsulfone, iodoacetamideor a maleimide moiety). In accordance with the present invention, usefulPEG includes substantially linear, straight chain PEG, branched PEG, ordendritic PEG. (See, e.g., Merrill, U.S. Pat. No. 5,171,264; Harris etal., Multiarmed, monofunctional, polymer for coupling to molecules andsurfaces, U.S. Pat. No. 5,932,462; Shen, N-maleimidyl polymerderivatives, U.S. Pat. No. 6,602,498).

Additionally, physiologically acceptable salts of the compounds of thisinvention are also encompassed herein. By “physiologically acceptablesalts” is meant any salts that are known or later discovered to bepharmaceutically acceptable. Some examples are: acetate;trifluoroacetate; hydrohalides, such as hydrochloride and hydrobromide;sulfate; citrate; maleate; tartrate; glycolate; gluconate; succinate;mesylate; besylate; and oxalate salts.

General Methodology

The present invention relates to a process for preparing apharmacologically active compound involving selecting at least oneinternal conjugation site of an Fc domain sequence. The conjugation sitemust be amenable to conjugation of an additional functional moiety by adefined conjugation chemistry through the side chain of an amino acidresidue at the conjugation site. Achieving highly selective,site-specific conjugation to Fc, in accordance with the presentinvention, requires consideration of a diverse variety of designcriteria. First, the conjugation partner, i.e., the additionalfunctional moiety (or moieties) of interest, and a preferred conjugationor coupling chemistry must be defined or predetermined. Functionalmoieties such as, but not limited to, proteins, peptides, polymers orother non-peptide organic moieties (e.g., “small molecules”), can beconjugated or coupled to the selected conjugation site through anassortment of different conjugation chemistries known in the art. Forexample, a maleimide-activated conjugation partner targeting anaccessible cysteine thiol on the Fc domain is one embodiment, butnumerous conjugation or coupling chemistries targeting the side chainsof either canonical or non-canonical, e.g., unnatural amino acids in theFc domain sequence, can be employed in accordance with the presentinvention.

Chemistries for the chemoselective conjugation, in accordance with thepresent invention, to specifically derivatized peptides, polymers, smallmolecules, or other agents to engineer proteins displaying novel andspecifically reactive side chain functionality include:copper(I)-catalyzed azide-alkyne[3+2] dipolar cycloadditions, Staudingerligation, other acyl transfers processes (S→N; X→N), oximations,hydrazone bonding formation and other suitable organic chemistryreactions such as cross-couplings using water-soluble palladiumcatalysts. (E.g., Bong et al., Chemoselective Pd(0)-catalyzed peptidecoupling in water, Organic Letters 3(16):2509-11 (2001); Dibowski etal., Bioconjugation of peptides by palladium-catalyzed C—Ccross-coupling in water, Angew. Chem. Int. Ed. 37(4):476-78 (1998);DeVasher et al., Aqueous-phase, palladium-catalyzed cross-coupling ofaryl bromides under mild conditions, using water-soluble, stericallydemanding alkylphosphines, J. Org. Chem. 69:7919-27 (2004); Shaugnessyet al., J. Org. Chem, 2003, 68, 6767-6774; Prescher, J A and Bertozzi CR, Chemistry in living system, Nature Chemical Biology 1(1); 13-21(2005)). Some useful conjugation chemistries are illustrated in Table 1Bbelow.

Table 1B.

Some useful conjugation chemistries. Citations: 17=Link et al.,Presentation and detection of azide functionality in bacterial cellsurface proteins, J. Am. Chem. Soc. 126:10598-602 (2004); 19=Chen etal., Site-specific labeling of cell surface proteins with biophysicalprobes using biotin ligase, Nat. Methods 2:99-104 (2005); 20=Zhang etal., A new strategy for the site-specific modification of proteins invivo, Biochemistry 42:6735-46 (2003); 22=Mahal et al., Engineeringchemical reactivity on cell surfaces through oligosaccharidebiosynthesis, Science 276:1125-28 (1997); 25=Kho et al., Atagging-via-substrate technology for detection and proteomics offarnesylated proteins, Proc. Natl. Acad. Sci. USA 101:12479-484 (2004);26=Speers et al., Activity-based protein profiling in vivo using acopper(I)-catalyzed azide-alkyne[3+2] cycloaddition, J. Am. Chem Soc.125:4686-87 (2003); 29=Speers et al., Profiling enzyme activities invivo using click chemistry, methods, Chem. Biol. 11:535-46 (2004);30=Prescher et al., Chemical remodeling of cell surfaces in livinganimals, Nature 430:873-77 (2004); 34=Agard et al., A strain-promoted[3+2] azide-alkyne cycloaddition for covalent modification ofbiomolecules in living systems, J. Am. Chem. Soc. 126:15046-47 (2004).

TABLE 1B

  Ketone/aldehyde

Protein¹⁹²⁰ Glycan²²

R—N₃ Azide Staudinger ligation  

Protein¹⁷²⁶ Glycan³⁰³⁴ Lipid²⁵ 'Click' Chemistry  

Strain-promoted cycloaddition  

'Click' chemistry  

Protein²⁹ Terminal alkyne

As mentioned above, the conjugation (or covalent binding) to the Fcdomain is through the side chain of an amino acid residue at theconjugation site, for example, but not limited to, a cysteinyl residue.The amino acid residue, for example, a cysteinyl residue, at theinternal conjugation site that is selected can be one that occupies thesame amino acid residue position in a native Fc domain sequence, or theamino acid residue can be engineered into the Fc domain sequence bysubstitution or insertion. Such amino acid residues can have either L orD stereochemistry (except for Gly, which is neither L nor D) and thepolypeptides, peptides and compositions of the present invention cancomprise a combination of stereochemistries. However, the Lstereochemistry is preferred. The invention also provides reversemolecules wherein the amino terminal to carboxy terminal sequence of theamino acids is reversed. For example, the reverse of a molecule havingthe normal sequence X₁-X₂-X₃ would be X₃-X₂-X₁. The invention alsoprovides retro-reverse molecules wherein, as above, the amino terminalto carboxy terminal sequence of amino acids is reversed and residuesthat are normally “L” enantiomers are altered to the “D” stereoisomerform.

Stereoisomers (e.g., D-amino acids) of the twenty canonical amino acids,and other non-canonical amino acids, described herein, such as unnaturalamino acids, can also be suitable components for polypeptide or peptideportions of certain embodiments of the inventive composition of matter.

Other examples of unnatural amino acid residues that can be particularlyuseful as the conjugation site in some embodiments of the inventiveprocesses and compositions of matter include: azido-containing aminoacid residues, e.g., azidohomoalanine, p-azido-phenylalanine;keto-containing amino acid residues, e.g., p-acetyl-phenylalanine;alkyne-containing amino acid residues, e.g., p-ethynylphenylalanine,homopropargylglycine, p-(prop-2-ynyl)-tyrosine; alkene-containing aminoacid residues e.g., homoallylglycine; aryl halide-containing amino acidresidues e.g. p-iodophenylalanine, p-bromophenylalanine; and1,2-aminothiol containing amino acid residues.

The non-canonical amino acid residues can be incorporated by amino acidsubstitution or insertion. Non-canonical amino acid residues can beincorporated into the peptide by chemical peptide synthesis rather thanby synthesis in biological systems, such as recombinantly expressingcells, or alternatively the skilled artisan can employ known techniquesof protein engineering that use recombinantly expressing cells. (See,e.g., Link et al., Non-canonical amino acids in protein engineering,Current Opinion in Biotechnology, 14(6):603-609 (2003); Schultz et al.,In vivo incorporation of unnatural amino acids, U.S. Pat. No.7,045,337).

The selection of the placement of the conjugation site in the overall Fcsequence is another important facet of selecting an internal conjugationsite in accordance with the present invention. Any of the exposed aminoacid residues on the Fc surface, or either of the Fc CH2 or CH3 loopregions or subdomains can be potentially useful conjugation sites(FIG. 1) and can be mutated to cysteine or some other reactive aminoacid for site-selective coupling, if not already present at the selectedconjugation site of the Fc domain sequence. However, this approach doesnot take into account potential steric constraints that may perturb theactivity of the fusion partner or limit the reactivity of the engineeredmutation. For example, a cysteine engineered to be fully solvent-exposedmay become oxidized during purification, leaving little or no reactivethiol for conjugation. Furthermore, the mutation introduced forconjugation, be it cysteine or any other amino acid, should notdestabilize the Fc structure or interfere with expression or recoveryyields of the Fc analog. Finally, in the case of an intact Fc domain,selected conjugation sites should be allosteric to the Fc dimerinterface, if present. Also, some therapeutic applications may furtherbenefit by maintaining conjugation sites distal to the Fc receptor(FcRn) interface.

In this invention a detailed topographical survey of the immunoglobulinFc surface structure is described, which identifies solvent exposedamino acids representing potentially suitable conjugation sites forchemically coupling proteins, peptides, polymers or other smallmolecules (FIG. 1). In this analysis, not all the hydrophilic,solvent-exposed residues were deemed suitable for conjugation. In fact,only 36 residues of a possible 115 were selected based on theirjuxtaposition with the FcRn binding and dimer interfaces as well asother localized steric constraints. The list of potential conjugationsites was further refined using the available Fc domain crystalstructures, their receptors and numerous Fc sequence alignments to mapall of the putative Fc structural loop regions (FIG. 2, boldface).Specific residues that are most suitable for substitution within theseloop region were identified by homology modeling and solventaccessibility (FIG. 2, underlined). Finally, each of these potentialconjugation sites were ranked based on their juxtaposition relative tothe FcRn and dimer interface, inter-species and isotype homologies andthe sites' proximity and involvement in key elements of Fc secondarystructure (Table 2). This approach using structure-based homologymodeling to identify Fc loop regions and to predict insertion-tolerantmutation sites has been previously validated using therapeutic peptideinsertions as described in Amgen patent application U.S. Prov. Appln.No. 60/612,680 filed Sep. 24, 2004. (See, WO 2006/036834) Based on thatwork, the most preferred mutation sites are Thr140, Asn78 and Glu50(FIG. 2; amino acid residue positions cited relate to reference sequenceSEQ ID NO:599).

To compare the preferred conjugation sites selected from the solventexposed surface residues highlighted in FIG. 1, with the boldfacedputative loop regions and the underlined preferred conjugation siteswithin those loops (FIG. 2), the two sequences were aligned and mappedto the human IgG1 Fc domain as shown in FIG. 3. Here emerges a veryconsistent agreement between the surface exposure model and the loopmodel for selecting potential conjugation sites. Clearly, these examplesdemonstrate that through a detailed structural analysis and comparisonof immunoglobulin Fc domains it is possible to identify anexperimentally manageable number of potential conjugation sites that arenot readily obvious from simple hydrophobic maps of the sequence.

Another subset of preferred mutations for coupling specificallyaddresses the use of cysteine analogs wherein the free thiolfunctionality must be preserved for efficient conjugation. This strategypresumes that cysteine mutations should be engineered into comparativelyrigid elements of secondary structure, as opposed to loop regions, andthe cysteine thiol should be juxtaposed within a pocket on the proteinsurface, providing minimal solvent exposure, to help protect it fromoxidation. This strategy has been effectively demonstrated in U.S. Pat.No. 6,420,339. Under this approach, the most preferred residues forcysteine mutation are, but not limited to, Ser196, Gln143, Leu139 andSer145 of the human Fc sequence (FIG. 4), with the positions recitedbeing relative to reference sequence SEQ ID NO: 599.

The inventors further envision as part of this invention that none ofthese potential conjugation sites require a full-length immunoglobulinFc domain to provide suitable substrates for coupling proteins,peptides, polymers, or other small molecules. In fact, any truncation ofFc that still includes a potential conjugation site recognized by thisinvention can be used for conjugation. For example, a CH2 subdomain orCH3 subdomain of an Fc greater than about 9 kD can be a useful “Fedomain” in accordance with the invention. Thus, this invention includesisolated Fc truncations, such as the CH2 or CH3 loop regions orsubdomains. Further, given the highly conserved three-dimensionalstructure of the “immunoglobulin fold” equivalent conjugation sites canbe readily deduced in other Ig Fc isotypes, truncations and subdomains,by sequence alignment and are therefore included in this invention.

Table 2 shows human Fc surface residues (using Protein Database file1FC1 as the data source) (S239 from the PDB file corresponds to S19 ofreference sequence SEQ ID NO:600 and S20 of reference sequence SEQ IDNO:599; K246 corresponds to K26 of SEQ ID NO:600 and K27 of SEQ IDNO:599, etc.).

TABLE 2 Human Fc surface residues, wherein 239S (i.e., S239) correspondsto S20 of reference sequence SEQ ID NO: 599. 239S 246K 248K* 249D 254S*255R* 256T* 258E 260T 265D+ 267S+ 268H+ 269E+ 270D 272E+ 274K+ 276N 278Y280D+ 281G+ 283Q 285H 286N 287A 288K* 289T 290K 292R 293E 294Q 295Q+296Y 297N+ 298S+ 299T 300Y 307T* 310H* 311Q* 312N 315D 316G 317K 318E+320K 322K 324S 326K+ 327A 330A+ 333E 334K 335T 337S 338K 339A 340K+341G+ 342Q+ 344R 345E 347Q 350T 354S 355R+ 356E+ 359T+ 360K+ 361N+ 362Q+371G 373Y 375S 376D 380E 382E* 383S 384N 385G* 386Q* 388E 389N+ 390N391Y 392K+ 393T 394T# 399D# 400S+ 401N 402G+ 403S 407Y# 409K# 411T 413D+414K+ 415S+ 416R+ 418Q+ 419Q+ 420G+ 421N+ 424S 430E 431A 433H* 434N*435H* 436Y* 437T 438Q* 439K 440S 442S+ *indicates likely FcRninteracting residues based on rat structures (not good candidates);#indicates dimer interaction domains (not good candidates); +indicatesbest candidates for modification.

Table 3 below shows prioritized sites for mutation or modification inthe predicted loop regions of human IgG1 Fc domain Amino acid residuepositions are numbered here in relation to reference sequence SEQ ID NO:599.

TABLE 3 Prioritized sites for mutation or modification in the predictedloop regions of human IgG1 Fc domain. Domain Loop Insertion CH2 D₄₆-E₅₃H₄₉/E₅₀ - 1^(st) E₅₀/D₅₁ - 2^(nd) CH2 E₇₄-T₈₀ Y₇₇/N₇₈ - 1^(st) N₇₈/S₇₉ -2^(nd) CH2-CH3 N₁₀₆-P₁₂₇ K₁₀₇/A₁₀₈ - 1^(st) linker N₁₀₆/K₁₀₇ - 2^(nd)CH3 D₁₃₇-K₁₄₁ L₁₃₉/T₁₄₀ - 1^(st) E₁₃₈/L₁₃₉ - 2^(nd) CH3 N₁₆₅-N₁₇₇E₁₆₉/N₁₇₀ - 1^(st) N₁₇₀/N₁₇₁ - 2^(nd) CH3 T₁₇₅-S₁₈₄ S₁₈₁/D₁₈₂ - 1^(st)V₁₇₈/L₁₇₉ - 2^(nd) CH3 K₁₉₅-V₂₀₃ G₂₀₁/N₂₀₂ - 1^(st) N₂₀₂/V₂₀₃ - 2^(nd)CH3 NA Q₁₆₇/P₁₆₈ CH3 NA G₁₈₃/S₁₈₄

In summary, this specification details a systematic approach to theidentification of useful conjugation sites on the surface ofimmunoglobulin Fc and includes all the mutation sites described herein.The identification of specific conjugation sites derives from theapplication of structural and sequence data to a detailed set ofstructure/function criteria developed by these inventors.

Structure of Inventive Compounds

Fc Domains.

This inventive composition requires the presence of at least one Fcdomain monomer, but multimeric Fc embodiments (e.g., Fc domain dimers,trimers, tetramers, pentamers, etc.) are also preferred. Both native Fcsand Fc variants are suitable Fc domains for use within the scope of thisinvention, as are Fc domains comprised in antibodies. A native Fc may beextensively modified to form an Fc variant in accordance with thisinvention, provided binding to the salvage receptor is maintained; see,for example WO 97/34631 and WO 96/32478. In some useful embodiments, onecan remove one or more sites of a native Fc that provide structuralfeatures or functional activity not required by a molecule of thisinvention, such as a fusion molecule. One may remove these sites by, forexample, substituting or deleting amino acid residues, insertingresidues into the site, or truncating portions containing the site. Theinserted or substituted residues may also be altered amino acids, suchas peptidomimetics or D-amino acids. Fc variants may be desirable for anumber of reasons, several of which are described below.

Exemplary Fc variants include molecules and sequences in which:

-   1. Sites involved in disulfide bond formation are removed. Such    removal may avoid reaction with other cysteine-containing proteins    present in the host cell used to produce the molecules of the    invention. For this purpose, the cysteine-containing segment at the    N-terminus may be truncated or cysteine residues may be deleted or    substituted with other amino acids (e.g., alanyl, seryl). For    example, one may truncate the N-terminal segment (truncations up to    about the first 20-amino acid residues of reference sequence SEQ ID    NO: 599 or SEQ ID NO:600) or delete or substitute the cysteine    residues at positions 7 and 10 of SEQ ID NO: 599 (positions 6 and 9    of SEQ ID NO:600). Even when cysteine residues are removed, the    single chain Fc domains can still form a dimeric Fc domain that is    held together non-covalently.-   2. A native Fc is modified to make it more compatible with a    selected host cell. For example, one may remove the PA sequence near    the N-terminus of a typical native Fc, which may be recognized by a    digestive enzyme in E. coli such as proline iminopeptidase. One may    also add an N-terminal methionine residue, especially when the    molecule is expressed recombinantly in a bacterial cell such as E.    coli. The Fc domain of reference sequence SEQ ID NO: 599 (FIG. 2) is    one such Fc variant, in which a methionine has been added to the    N-terminal of SEQ ID NO: 600.-   3. A portion of the N-terminus of a native Fc is removed to prevent    N-terminal heterogeneity when expressed in a selected host cell. For    this purpose, one may delete any or all of the first 20 amino acid    residues at the N-terminus, particularly those corresponding to    positions 1, 2, 3, 4 and 5 of reference sequence SEQ ID NO: 600.-   4. One or more glycosylation sites are removed. Residues that are    typically glycosylated (e.g., asparagine) may confer cytolytic    response. Such residues may be deleted or substituted with    unglycosylated residues (e.g., alanine).-   5. Sites involved in interaction with complement, such as the Clq    binding site, are removed. For example, one may delete or substitute    the EKK sequence of human IgG1. Complement recruitment may not be    advantageous for the molecules of this invention and so may be    avoided with such an Fc variant.-   6. Sites are removed that affect binding to Fc receptors other than    a salvage receptor. A native Fc may have sites for interaction with    certain white blood cells that are not required for the fusion    molecules of the present invention and so may be removed.-   7. The ADCC site is removed. ADCC sites are known in the art; see,    for example, Molec. Immunol 29 (5): 633-9 (1992) with regard to ADCC    sites in IgG1. These sites, as well, are not required for the fusion    molecules of the present invention and so may be removed.-   8. When the native Fc is derived from a non-human antibody, the    native Fc may be humanized. Typically, to humanize a native Fc, one    will substitute selected residues in the non-human native Fc with    residues that are normally found in human native Fc. Techniques for    antibody humanization are well known in the art.    Preferred Fc variants include the following. In reference sequence    SEQ ID NO: 599 (FIG. 2) the leucine at position 15 may be    substituted with glutamate; the glutamate at position 99, with    alanine; and the lysines at positions 101 and 103, with alanines. In    addition, one or more tyrosine residues can be replaced by    phenyalanine residues.

In some preferred embodiments, the Fc domain is an IgG1 Fc domaincomprising an amino acid sequence SEQ ID NO: 603:

(SEQ ID NO: 603) Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro CysPro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys ProLys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val AspVal Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val GluVal His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr ArgVal Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu TyrLys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile SerLys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser ArgAsp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe TyrPro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn TyrLys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser LysLeu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser ValMet His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser ProGly Lys//;andthe one or more specifically selected conjugation site(s) is selectedfrom an amino acid residue position contained in a loop region thatcomprises an amino acid sequence selected from the group consisting of

SEQ ID NO: 601 Pro Pro //, SEQ ID NO: 602 Asp Val Ser His Glu Asp ProGlu//, SEQ ID NO: 604 Val His Asn Ala//, SEQ ID NO: 605 Glu Glu Gln TyrAsn Ser Thr//, SEQ ID NO: 606 Val Leu His Gln Asp Trp Leu Asn Gly LysGlu//, SEQ ID NO: 607 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr IleSer Lys Ala Lys Gly Gln Pro Arg Glu Pro//, SEQ ID NO: 608 Asp Glu LeuThr Lys//, SEQ ID NO: 609 Asn Gly Gln Pro Glu Asn Asn//, SEQ ID NO: 610Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser//,and

SEQ ID NO: 611 Lys Ser Arg Trp Gln Gln Gly Asn Val//.

In the compositions of matter prepared in accordance with thisinvention, at least one additional functional moiety is covalently boundto a monomeric or multimeric Fc domain through a specifically selectedconjugation site involving an amino acid residue side chain selected asdescribed herein. Optionally, other moieties, such as a polypeptide,peptide, peptidomimetic or non-peptide organic moiety can be attached tothe Fc domain through the Fc domain's N-terminus (i.e., via the α-aminosite) or C-terminus (i.e., via the α-carboxy site).

Certain embodiments of the molecules of this invention may be describedby the following formula I:

wherein:

-   -   F¹ is a monomer of the monomeric or multimeric Fc domain;    -   X¹ is covalently bound to the N-terminus of F¹ through the        α-amino site of F¹;    -   X² is covalently bound to the C-terminus of F¹ through the        α-carboxy site of F¹;    -   X³ is covalently bound to the one or more specifically selected        conjugation site(s) in F¹ selected from the group consisting of        underlined residue positions in FIG. 1, boldface residue        positions in FIG. 2, highlighted residue positions in FIG. 3,        underlined residue positions in FIG. 3, and a cysteine residue        added to the Fc domain by substitution at an Fc site selected        from the group consisting of Leu139, Gln143, Ser145, and Ser196,        or, if g>1, any combination of these members;    -   X¹, X², and X³ are each independently selected from        -(L¹)_(c)-P⁰, -(L¹)_(c)-P¹, (L¹)_(c)-P¹-(L²)_(d)-P²,        (L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(c)-P³, and        -(L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³-(L⁴)_(f)-P⁴;    -   P⁰, P¹, P², P³, and P⁴ are each independently selected from the        group consisting of:        -   i) a pharmaceutically acceptable polymer or dextran;        -   ii) a pharmacologically active polypeptide, peptide,            peptidomimetic, or non-peptide organic moiety;        -   iii) a radioisotope, an enzyme, a biotinyl moiety, a            fluorophore, or a chromophore; and        -   iv) an immobilized substrate, provided that in a chain            comprising more than one additional functional moieties, the            immobilized substrate is the moiety most distal from F¹, and            there can be no more than one immobilized substrate in the            chain;    -   L¹, L², L³, and L⁴ are each independently linkers;    -   a, b, c, d, e, and f are each independently 0 or 1; and        g is 1, 2, 3, or 4.

Those of ordinary skill in the art will appreciate that more than oneadditional functional moieties (X³) can be attached to the Fc domain,and that the multiple X³ substituents may be the same or different; forexample, comprising same or different P¹ functional moiety (i.e., a P¹in a given formula may be the same or different from any other P¹, P²,P³, or P⁴), different linkers attached to the same peptide sequence, andso on. Likewise, X¹ and X² may be the same, different, or absent (i.e.,a and/or b=0), and the integers c through f may be different for X¹, X²,and X³.

Thus, compounds of Formula I encompass, but are not limited to,exemplary embodiments of the inventive compounds of the followingformulae (II)-(XXVIII):

and multimers thereof, wherein a=1, b=0, F¹ is attached at theC-terminus of a polypeptide or peptide comprised in X¹, and X³ isattached through a specifically selected internal conjugation site inF¹;

and multimers thereof, wherein a=0, b=1, F¹ is attached at theN-terminus of a polypeptide or peptide comprised in X², and X³ isattached through a specifically selected internal conjugation site inF¹;

and multimers thereof, wherein a=0, b=1, F¹ is attached through theN-terminus of a polypeptide or peptide P¹ comprised in -(L¹)_(c)-P¹ and-(L¹)_(c)-PEG is attached through a specifically selected internalconjugation site in F¹;

and multimers thereof, wherein a=0, b=1, F¹ is attached through theN-terminus of a polypeptide or peptide P¹ comprised in-(L¹)_(c)-P¹-(L²)_(d)-P² and (L¹)_(c)-PEG is attached through aspecifically selected internal conjugation site in F¹;

and multimers thereof, wherein a=0, b=1, F¹ is attached through theN-terminus of a polypeptide or peptide P⁰ comprised in -(L¹)_(c)-P⁰ and-(L¹)_(c)-PEG is attached through a specifically selected internalconjugation site in F¹;

and multimers thereof, wherein a=0, b=1, F¹ is attached through theN-terminus of a polypeptide or peptide P⁰ comprised in -(L¹)_(c)-P⁰ and-(L¹)_(c)-P¹ is attached through a specifically selected internalconjugation site in F¹;

and multimers thereof, wherein a=0, b=1, F¹ is attached through theN-terminus of a polypeptide or peptide P⁰ comprised in -(L¹)_(c)-P⁰ and-(L¹)_(c)-P¹-(L²)_(d)-P² is attached through a specifically selectedinternal conjugation site in F¹;

and multimers thereof, wherein a=0, b=1, g=2, F¹ is attached through theN-terminus of a polypeptide or peptide P⁰ comprised in -(L¹)_(c)-P⁰, and-(L¹)_(c)-PEG and -(L¹)_(c)-P¹ are each independently attached throughspecifically selected internal conjugation sites in F¹;

and multimers thereof, wherein a=0, b=1, g=2, F¹ is attached through theN-terminus of a polypeptide or peptide P⁰ comprised in -(L¹)_(c)-P⁰ and-(L¹)_(c)-PEG and -(L¹)_(c)-P¹-(L²)_(d)-P² are each independentlyattached through specifically selected internal conjugation sites in F¹;

and multimers thereof, wherein a=0, b=1, F¹ is attached at theN-terminus of a polypeptide or peptide —P⁰, and -(L¹)_(c)-PEG and-(L¹)_(c)-P²-(L²)_(d)-P² are each independently attached throughspecifically selected internal conjugation sites in F¹.

and multimers thereof, wherein a=1, b=0, F¹ is attached through theC-terminus of a polypeptide or peptide P¹ comprised in -(L¹)_(c)-P¹ and-(L¹)_(c)-PEG is attached through a specifically selected internalconjugation site in F¹;

and multimers thereof, wherein a=1, b=0, F¹ is attached through theC-terminus of a polypeptide or peptide P¹ comprised in-(L¹)_(c)-P¹-(L²)_(d)-P² and (L¹)_(c)-PEG is L attached through aspecifically selected internal conjugation site in F¹;

and multimers thereof, wherein a=1, b=0, F¹ is attached through theC-terminus of a polypeptide or peptide P⁰ comprised in -(L¹)_(c)-P⁰ and-(L¹)_(c)-PEG is attached through a specifically selected internalconjugation site in F¹;

and multimers thereof, wherein a=1, b=0, F¹ is attached through theC-terminus of a polypeptide or peptide P⁰ comprised in -(L¹)_(c)-P⁰ and(L¹)_(c)-P¹ is attached through a specifically selected internalconjugation site in F¹;

and multimers thereof, wherein a=1, b=0, F¹ is attached through theC-terminus of a polypeptide or peptide P⁰ comprised in -(L¹)_(c)-P⁰, and-(L¹)_(c)-P¹-(L²)_(d)-P² is attached through a specifically selectedinternal conjugation site in F¹;

and multimers thereof, wherein a=1, b=0, g=2, F¹ is attached through theC-terminus of a polypeptide or peptide P⁰ comprised in -(L¹)_(c)-P⁰ and(L¹)_(c)-PEG and (L¹)_(c)-P¹ are each independently attached throughspecifically selected internal conjugation sites in F¹;

and multimers thereof, wherein a=1, b=0, g=2, F¹ is attached at theC-terminus of a polypeptide or peptide —P⁰ and -(L¹)_(c)-PEG and-(L¹)_(c)-P¹-(L²)_(d)-P² are each independently attached throughspecifically selected internal conjugation sites in F¹;

and multimers thereof, wherein a=1, b=0, g=2, F¹ is attached through theC-terminus of a polypeptide or peptide P¹ comprised in -(L¹)_(c)-P¹, and-(L¹)_(c)-PEG and -(L¹)_(c)-P²-(L²)_(d)-P² are each independentlyattached through specifically selected internal conjugation sites in F¹;

and multimers thereof, wherein a=1, b=1, P⁰-(L¹)_(c)-F¹-(L¹)_(c)-P¹ isattached as written from the N-terminus of a polypeptide or peptide P⁰to the C-terminus of a polypeptide or peptide P¹ and -(L¹)_(c)-PEG isattached through a specifically selected internal conjugation site inF¹;

and multimers thereof, wherein a=1, b=1, c=1,P⁰-(L¹)_(c)-F¹-(L¹)_(c)-P¹-(L²)_(d)-P² is attached as written from theN-terminus of a polypeptide or peptide P⁰ to the C-terminus of apolypeptide or peptide P¹ (if P¹ but not P² is a polypeptide or peptide)or P² (if both P¹ and P² are a polypeptide or peptide) and -(L¹)_(c)-PEGis attached through a specifically selected internal conjugation site inF¹;

and multimers thereof, wherein a=1, b=1, P⁰—F¹-(L¹)_(c)-P¹-(L²)_(d)-P²is attached as written from the N-terminus of a polypeptide or peptideP⁰ to the C-terminus of a polypeptide or peptide P¹ (if P¹ but not P² isa polypeptide or peptide) or P² (if both P¹ and P² are a polypeptide orpeptide), and -(L¹)_(c)-PEG is attached through a specifically selectedinternal conjugation site in F¹;

and multimers thereof, wherein a=1, b=1, c=1, P¹-(L¹)_(c)-F¹-(L¹)_(c)-P⁰is attached as written from the N-terminus of a polypeptide or peptideP¹ to the C-terminus of a polypeptide or peptide P⁰ and -(L¹)_(c)-PEG isattached through a specifically selected internal conjugation site inF¹;

and multimers thereof, wherein a=1, b=1, c=1, P¹-(L¹)_(c)-F¹-(L¹)_(c)-P⁰is attached as written from the N-terminus of a polypeptide or peptideP¹ to the C-terminus of a polypeptide or peptide P⁰ and (L¹)_(c)-P¹ isattached through a specifically selected internal conjugation site inF¹;

and multimers thereof, wherein a=1, b=1, c=1, P¹-(L¹)_(c)-F¹-(L¹)_(c)-P⁰is attached as written from the N-terminus of a polypeptide or peptideP¹ to the C-terminus of a polypeptide or peptide P⁰, and-(L¹)_(c)-P¹-(L²)_(d)-P² is attached through a specifically selectedinternal conjugation site in F¹;

and multimers thereof, wherein a=1, b=1, c=1, P¹-(L¹)_(c)-F¹-(L¹)_(c)-P⁰is attached as written from the N-terminus of a polypeptide or peptideP¹ to the C-terminus of a polypeptide or peptide P⁰ and -(L¹)_(c)-PEGand -(L¹)_(c)-P¹ are each independently attached through specificallyselected internal conjugation sites in F¹;

and multimers thereof, wherein a=1, b=1, c=1, P¹-(L¹)_(c)-F¹-(L¹)_(c)-P⁰is attached as written from the N-terminus of a polypeptide or peptideP¹ to the C-terminus of a polypeptide or peptide P⁰, and -(L¹)_(c)-PEGand -(L¹)_(c)-P¹-(L²)_(d)-P² are each independently attached throughspecifically selected internal conjugation sites in F¹; and

and multimers thereof, wherein a=1, b=1, c=1, P¹-(L¹)_(c)-F¹-(L¹)_(c) P⁰is attached as written from the N-terminus of a polypeptide or peptideP¹ to the C-terminus of a polypeptide or peptide P⁰, and the second-(L¹)_(c)-P¹ and (L¹)_(c)-P²-(L²)_(d)-P² are each independently attachedthrough specifically selected internal conjugation sites in F¹.

In another embodiment of the present invention, the composition ofmatter is an antibody modified, which comprises at least one additionalfunctional moiety (X³) covalently bound to the Fc domain of the antibodythrough one or more specifically selected conjugation site(s) in the Fcdomain. The conjugation site, or sites, are selected from: underlinedresidue positions in FIG. 1, boldface residue positions in FIG. 2,highlighted residue positions in FIG. 3, underlined residue positions inFIG. 3, or a cysteine residue added to the Fc domain by substitution atan Fc site selected from the group consisting of Leu139, Gln143, Ser145,and Ser196, or, if there is more than one X³, any combination of thesemembers. X³ is selected from -(L¹)_(c)-P⁰, -(L¹)_(c) P¹, (L¹)_(c)P¹-(L²)_(d)-P², -(L¹)_(c) P¹-(L²)_(d)-P²-(L³)_(e)-P³, and-(L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³-(L⁴)_(f)-P⁴;

-   -   P⁰, P¹, P², P³, and P⁴ are each independently selected from the        group consisting of:        -   i) a pharmaceutically acceptable polymer or dextran;        -   ii) a pharmacologically active polypeptide, peptide,            peptidomimetic, or non-peptide organic moiety;        -   iii) a radioisotope, an enzyme, a biotinyl moiety, a            fluorophore, or a chromophore; and        -   iv) an immobilized substrate, provided that in a chain            comprising more than one additional functional moieties, the            immobilized substrate is the moiety most distal from the Fc            domain, and there can be no more than one immobilized            substrate in the chain;    -   L¹, L², L³, and L⁴ are each independently linkers;    -   c, d, e, and f are each independently 0 or 1.

Additional functional moiety or moieties (X³)

Some embodiments of the additional functional moiety, or moieties,(i.e., P⁰, P¹, P², P³, P⁴, that can be conjugated to the Fc domain, inaccordance with the present invention, will now be exemplified ingreater detail.

Polypeptides or Peptides.

One or more additional functional moieties is conjugated to the Fcdomain molecules of this invention. Such additional functional moietiescan include a polypeptide, a peptide, an antibody, antibody fragment,(or a non-peptide organic molecule “small molecules”, e.g., apeptidomimetic compound) capable of binding to a salvage receptor. Forexample, one can use as a functional moiety a polypeptide as describedin U.S. Pat. No. 5,739,277, issued Apr. 14, 1998 to Presta et al.Peptides of interest can also be selected by phage display for bindingto the FcRn salvage receptor. Such salvage receptor-binding compoundsare also included within the meaning of “functional moiety” in thisinvention. Such functional moieties can be selected, for example, forincreased half-life (e.g., by avoiding sequences recognized byproteases) and decreased immunogenicity (e.g., by favoringnon-immunogenic sequences, as discovered in antibody humanization).

In other embodiments, a variety of other peptides or polypeptides can beused as the additional functional moiety in conjunction with the presentinvention. Exemplary polypeptides that can be used include thosementioned as fusion partners in Table 1 hereinabove. Preferredpolypeptides have therapeutic utility and include the human proteinsanakinra, sTNF-R2, sTNF-R1, CTLA4, OPG, GDNF, PTH fragments, glucagonsfragments, GLP-1, and the like. Accordingly, a preferred polypeptidesequence is the sTNF-R2 sequence below:

(SEQ ID NO: 617) QICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGDFALPVGLIVGVTALGLLIIGVVNCVIMTQVKKKPLCLQREAKVPHLPADKARGTQGPEQQHLLITAPSSSSSSLESSASALDRRAPTRNQPQAPGVEASGAGEARASTGSSDSSPGGHGTQVNVTCIVNVCSSSDHSSQCSSQASSTMGDTDSSPSESPKDEQVPFSKEECAFRSQLETPETLLGSTEEKPLPLGVPDAGMKPS//

In accordance with the present invention, one may modify Fc fusionproteins comprising such polypeptides by adding an additional functionalmoiety such as PEG through a selected site in the Fc domain. In thisway, for example, a PEGylated derivative of etanercept is within thescope of this invention in which the PEG molecule is attached through aselected site in the Fc domain of etanercept. Such a molecule can bedescribed by formula XIV above in which P⁰-(L¹)_(c)-F¹ encodesetanercept (wherein P⁰ is SEQ ID NO: 618:

SEQ ID NO: 618 1 Leu-Pro-Ala-Gln-Val-Ala-Phe-Thr-Pro-Tyr- 11Ala-Pro-Glu-Pro-Gly-Ser-Thr-Cys-Arg-Leu- 21Arg-Glu-Tyr-Tyr-Asp-Gln-Thr-Ala-Gln-Met- 31Cys-Cys-Ser-Lys-Cys-Ser-Pro-Gly-Gln-His- 41Ala-Lys-Val-Phe-Cys-Thr-Lys-Thr-Ser-Asp- 51Thr-Val-Cys-Asp-Ser-Cys-Glu-Asp-Ser-Thr- 61Tyr-Thr-Gln-Leu-Trp-Asn-Trp-Val-Pro-Glu- 71Cys-Leu-Ser-Cys-Gly-Ser-Arg-Cys-Ser-Ser- 81Asp-Gln-Val-Glu-Thr-Gln-Ala-Cys-Thr-Arg- 91Glu-Gln-Asn-Arg-Ile-Cys-Thr-Cys-Arg-Pro- 101Gly-Trp-Tyr-Cys-Ala-Leu-Ser-Lys-Gln-Glu- 111Gly-Cys-Arg-Leu-Cys-Ala-Pro-Leu-Arg-Lys- 121Cys-Arg-Pro-Gly-Phe-Gly-Val-Ala-Arg-Pro- 131Gly-Thr-Glu-Thr-Ser-Asp-Val-Val-Cys-Lys- 141Pro-Cys-Ala-Pro-Gly-Thr-Phe-Ser-Asn-Thr- 151Thr-Ser-Ser-Thr-Asp-Ile-Cys-Arg-Pro-His- 161Gln-Ile-Cys-Asn-Val-Val-Ala-Ile-Pro-Gly- 171Asn-Ala-Ser-Met-Asp-Ala-Val-Cys-Thr-Ser- 181Thr-Ser-Pro-Thr-Arg-Ser-Met-Ala-Pro-Gly- 191Ala-Val-His-Leu-Pro-Gln-Pro-Val-Ser-Thr- 201Arg-Ser-Gln-His-Thr-Gln-Pro-Thr-Pro-Glu- 211Pro-Ser-Thr-Ala-Pro-Ser-Thr-Ser-Phe-Leu- 221Leu-Pro-Met-Gly-Pro-Ser-Pro-Pro-Ala-Glu- 231Gly-Ser-Thr-Gly-Asp-Glu-Pro-Lys-Ser-Cys- 241Asp-Lys-Thr-His-Thr-Cys-Pro-Pro-Cys-Pro- 251Ala-Pro-Glu-Leu-Leu-Gly-Gly-Pro-Ser-Val- 261Phe-Leu-Phe-Pro-Pro-Lys-Pro-Lys-Asp-Thr- 271Leu-Met-Ile-Ser-Arg-Thr-Pro-Glu-Val-Thr- 281Cys-Val-Val-Val-Asp-Val-Ser-His-Glu-Asp- 291Pro-Glu-Val-Lys-Phe-Asn-Trp-Tyr-Val-Asp- 301Gly-Val-Glu-Val-His-Asn-Ala-Lys-Thr-Lys- 311Pro-Arg-Glu-Glu-Gln-Tyr-Asn-Ser-Thr-Tyr- 321Arg-Val-Val-Ser-Val-Leu-Thr-Val-Leu-His- 331Gln-Asp-Trp-Leu-Asn-Gly-Lys-Glu-Tyr-Lys- 341Cys-Lys-Val-Ser-Asn-Lys-Ala-Leu-Pro-Ala- 351Pro-Ile-Glu-Lys-Thr-Ile-Ser-Lys-Ala-Lys- 361Gly-Gln-Pro-Arg-Glu-Pro-Gln-Val-Tyr-Thr- 371Leu-Pro-Pro-Ser-Arg-Glu-Glu-Met-Thr-Lys- 381Asn-Gln-Val-Ser-Leu-Thr-Cys-Leu-Val-Lys- 391Gly-Phe-Tyr-Pro-Ser-Asp-Ile-Ala-Val-Glu- 401Trp-Glu-Ser-Asn-Gly-Gln-Pro-Glu-Asn-Asn- 411Tyr-Lys-Thr-Thr-Pro-Pro-Val-Leu-Asp-Ser- 421Asp-Gly-Ser-Phe-Phe-Leu-Tyr-Ser-Lys-Leu- 431Thr-Val-Asp-Lys-Ser-Arg-Trp-Gln-Gln-Gly- 441Asn-Val-Phe-Ser-Cys-Ser-Val-Met-His-Glu- 451Ala-Leu-His-Asn-His-Tyr-Thr-Gln-Lys-Ser- 461 Leu-Ser-Leu-Ser-Pro-Gly-Lys//,and c is 0) or a molecule based on the etanercept sequence with one ormore modified residues to enable linkage to the (L¹)_(c)-PEGsubstituent.

Also in accordance with this invention, a peptide or additionalpolypeptide functional moiety can be linked through a selected site inthe Fc domain. Alternatively, the polypeptide-Fc fusion protein can belinked to a peptide or tandem dimer, trimer, or tetramer (i.e.,-(L¹)_(c) P¹, (L¹)_(c)-P¹-(L²)_(d)-P²,(L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³, and-(L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³-(L⁴)_(f)-P⁴). The peptides can belinked through a selected internal Fc site or at an available N- orC-terminus of the fusion protein. In this way, this inventionencompasses an etanercept derivative comprising, for example, aBAFF-binding peptide dimer (see Table 10 hereinafter), a PEG moiety, orboth. In such a molecule for example, the structure can follow formulaXXI above wherein P⁰ is SEQ ID NO: 617, P¹ and P² are BAFF-bindingpeptides such as LPGCKWDLLIKQWVCDPL (SEQ ID NO: 514).

Any number of peptides or polypeptides can be used in conjunction withthe present invention. In some embodiments, the peptides or polypeptidesbind to angiopoietin-2 (ang-2), myostatin, nerve growth factor (NGF),tumor necrosis factor (TNF), B cell activating factor (BAFF, alsoreferred to as TALL-1) or mimic the activity of EPO, TPO, or G-CSF.Targeting peptides are also of interest, including tumor-homingpeptides, membrane-transporting peptides, and the like. All of theseclasses of peptides or polypeptides can be discovered by methodsdescribed in the references cited in this specification and otherreferences.

As mentioned above, phage display is useful in generating peptides foruse in the present invention. It has been stated that affinity selectionfrom libraries of random peptides can be used to identify peptideligands for any site of any gene product. Dedman et al. (1993), J. Biol.Chem. 268: 23025-30. Phage display is particularly well suited foridentifying peptides that bind to such proteins of interest as cellsurface receptors or any proteins having linear epitopes. Wilson et al.(1998), Can. J. Microbiol. 44: 313-29; Kay et al. (1998), Drug Disc.Today 3: 370-8. Such proteins are extensively reviewed in Herz et al.(1997), J. Receptor & Signal Transduction Res. 17(5): 671-776, which ishereby incorporated by reference. Such proteins of interest arepreferred for use in this invention.

A particularly preferred group of peptides are those that bind tocytokine receptors. Cytokines have recently been classified according totheir receptor code. See Inglot (1997), Archivum Immunologiae etTherapiae Experimentalis 45: 353-7, which is hereby incorporated byreference. Among these receptors, most preferred are the CKRs (family Iin Table 4). The receptor classification appears in Table 4.

TABLE 4 Cytokine Receptors Classified by Receptor Code Cytokines(ligands) Receptor Type Family Subfamily family subfamily I. 1. IL-2,IL-4, I. Cytokine R 1. shared γCr, Hematopoietic IL-7, IL-9, (CKR)IL-9R, IL-4R cytokines IL-13, IL-15 2. shared GP 2. IL-3, IL-5, 140 βRGM-CSF 3. 3.shared RP 3. IL-6, IL-11, 130, IL-6 R, IL-12, LIF, Leptin ROSM, CNTF, 4. “single chain” Leptin (OB) R, GCSF-R, 4. G-CSF, EPO,TPO-R, GH-R TPO, PRL, 5. other R^(a) GH 5. IL-17, HVS- IL-17 II. IL-10IL-10, BCRF-1, II. IL-10 R ligands HSV-IL-10 III. Interferons 1. IFN-α1,α2, III. Interferon R 1. IFNAR α4, m, t, IFN- 2. IFNGR β^(b) 2. IFN-γIV. IL-1 and 1. IL-1α, IL-1β, IV. IL-1R 1. IL-1R, IL- IL-1 like IL-1Ra1RAcP ligands 2. IL-18, IL- 2. IL-18R, IL- 18BP 18RAcP V. TNF family 1.TNF-α, 3. NGF/TNF R^(c) TNF-RI, AGP- TNF-β (LT), 3R, DR4, DR5, FASL,CD40 OX40, OPG, L, CD30L, TACI, CD40, CD27 L, FAS, ODR OX40L, OPGL,TRAIL, APRIL, AGP-3, BLys, TL5, Ntn-2, KAY, Neutrokine-α VI. 1. α 4.Chemokine R 1. CXCR Chemokines chemokines: 2. CCR IL-8, GRO α, 3. CR β,γ, IF-10, 4. DARC^(d) PF-4, SDF-1 2. β chemokines: MIP1α, MIP1β, MCP-1,2,3,4, RANTES, eotaxin 3. γ chemokines: lymphotactin VII. Growth 1.1SCF, M-CSF, VII. RKF 1. TK sub- factors PDGF-AA, family AB, BB, 1.1 IgTKIII R, KDR, FLT-1, VEGF-RI, FLT-3L, VEGF-RII VEGF, SSV- 1.2 IgTK IV RPDGF, HGF, 1.3 Cysteine-rich SF TK-I 1.2 FGFα, FGFβ 1.4 Cysteine rich1.3 EGF, TGF-α, TK-II, IGF- VV-F19 RI (EGF-like) 1.5 Cysteine knot 1.4IGF-I, IGF-II, TK V Insulin 2. Serine- 1.5 NGF, BDNF, threonine NT-3,NT-4^(e) kinase 2. TGF-β1,β2,β3 subfamily (STKS)^(f) ²Other IFN type Isubtypes remain unassigned. Hematopoietic cytokines, IL-10 ligands andinterferons do not possess functional intrinsic protein kinases. Thesignaling molecules for the cytokines are JAK's, STATs and relatednon-receptor molecules. IL-14, IL-16 and IL-18 have been cloned butaccording to the receptor code they remain unassigned. ²Other IFN type Isubtypes remain unassigned. Hematopoietic cytokines, IL-10 ligands andinterferons do not possess functional intrinsic protein kinases. Thesignaling molecules for the cytokines are JAK's, STATs and relatednon-receptor molecules. IL-14, IL-16 and IL-18 have been cloned butaccording to the receptor code they remain unassigned. ³TNF receptorsuse multiple, distinct intracellular molecules for signal transductionincluding “death domain” of FAS R and 55 kDa TNF-R that participates intheir cytotoxic effects. NGF/TNF R can bind both NGF and related factorsas well as TNF ligands. Chemokine receptors are seven transmembrane(7TM, serpentine) domain receptors. They are G protein-coupled. ⁴TheDuffy blood group antigen (DARC) is an erythrocyte receptor that canbind several different chemokines. IL-1R belongs to the immunoglobulinsuperfamily but their signal transduction events characteristics remainunclear. ⁵The neurotrophic cytokines can associate with NGF/TNFreceptors also. ⁶STKS may encompass many other TGF-β-related factorsthat remain unassigned. The protein kinases are intrinsic part of theintracellular domain of receptor kinase family (RKF). The enzymesparticipate in the signals transmission via the receptors.

Particular proteins of interest as targets for peptide generation in thepresent invention include, but are not limited to, the following:

-   -   αvβ3    -   αVβ1    -   Ang-2    -   BAFF/TALL-1    -   B7    -   B7RP1    -   CRP1    -   Calcitonin    -   CD28    -   CETP    -   cMet    -   Complement factor B    -   C4b    -   CTLA4    -   Glucagon    -   Glucagon Receptor    -   LIPG    -   MPL    -   myostatin    -   splice variants of molecules preferentially expressed on tumor    -   cells; e.g., CD44, CD30    -   unglycosylated variants of mucin and Lewis Y surface    -   glycoproteins    -   CD19, CD20, CD33, CD45    -   prostate specific membrane antigen and prostate specific cell        antigen    -   matrix metalloproteinases (MMPs), both secreted and        membrane-bound (e.g., MMP-9)    -   Cathepsins    -   angiopoietin-2    -   TIE-2 receptor    -   heparanase    -   urokinase plasminogen activator (UPA), UPA receptor    -   parathyroid hormone (PTH), parathyroid hormone-related protein        (PTHrP), PTH-RI, PTH-RII    -   Her2    -   Her3    -   Insulin

Exemplary peptides for this invention appear in Tables 4 through 20 ofU.S. Pat. No. 6,660,843, which are hereby incorporated by reference.Additional preferred peptides appear in U.S. 2003/0229023, publishedDec. 11, 2003; WO 03/057134, published Jul. 17, 2003; U.S. 2003/0236193,published Dec. 25, 2003; WO 00/24770, published May 4, 2000; U.S.2003/0176352, published Sep. 18, 2003; WO 03/031589, published Apr. 17,2003; U.S. Ser. No. 10/666,480, filed Sep. 18, 2003; WO 04/026329,published Apr. 1, 2004; U.S. Ser. No. 10/742,379, filed Dec. 19, 2003;PCT/US03/40781, filed Dec. 19, 2003, each of which are herebyincorporated by reference. Such peptides may be prepared by methodsdisclosed in the art.

The amino acid sequences of some preferred peptides and polypeptidesappear in Tables 5-10 below. Single letter amino acid abbreviations areused. Any of these peptides may be linked in tandem (i.e.,sequentially), with or without linkers. Any peptide containing acysteinyl residue may be cross-linked with another Cys-containingpeptide or protein. Any peptide having more than one Cys residue mayform an intrapeptide disulfide bond, as well. Any of these peptides maybe derivatized as described herein. All peptides are linked throughpeptide bonds unless otherwise noted.

TABLE 5 EPO-mimetic peptide sequences SEQUENCE SEQ ID NO:YXCXXGPXTWXCXP, wherein X is any amino 1 acid GGTYSCHFGPLTWVCKPQGG 2GGDYHCRMGPLTWVCKPLGG 3 GGVYACRMGPITWVCSPLGG 4 VGNYMCHFGPITWVCRPGGG 5GGLYLCRFGPVTWDCGYKGG 6 GGTYSCHFGPLTWVCKPQGGSSK 7 GGTYSCHGPLTWVCKPQGG 8VGNYMAHMGPITWVCRPGG 9 GGPHHVYACRMGPLTWIC 10 GGTYSCHFGPLTWVCKPQ 11GGLYACHMGPMTWVCQPLRG 12 TIAQYICYMGPETWECRPSPKA 13 YSCHFGPLTWVCK 14YCHFGPLTWVC 15 GGLYLCRFGPVTWDCGYKGG 16 GGTYSCHFGPLTWVCKPQGG 17GGDYHCRMGPLTWVCKPLGG 18 VGNYMCHFGPITWVCRPGGG 19 GGVYACRMGPITWVCSPLGG 20VGNYMAHMGPITWVCRPGG 21 GGTYSCHFGPLTWVCKPQ 22 GGLYACHMGPMTWVCQPLRG 23TIAQYICYMGPETWECRPSPKA 24 YSCHFGPLTWVCK 25 YCHFGPLTWVC 26 SCHFGPLTWVCK27

TABLE 6 TPO-mimetic peptide sequences SEQUENCE SEQ ID NO: IEGPTLRQWLAARA28 IEGPTLRQWLAAKA 29 IEGPTLREWLAARA 30 TLREWL 31 GRVRDQVAGW 32GRVKDQIAQL 33 GVRDQVSWAL 34 ESVREQVMKY 35 SVRSQISASL 36 GVRETVYRHM 37GVREVIVMHML 38 GRVRDQIWAAL 39 AGVRDQILIWL 40 GRVRDQIMLSL 41 CTLRQWLQGC42 CTLQEFLEGC 43 CTRTEWLHGC 44 CTLREWLHGGFC 45 CTLREWVFAGLC 46CTLRQWLILLGMC 47 CTLAEFLASGVEQC 48 CSLQEFLSHGGYVC 49 CTLREFLDPTTAVC 50CTLKEWLVSHEVWC 51 REGPTLRQWM 52 EGPTLRQWLA 53 ERGPFWAKAC 54 REGPRCVMWM55 CGTEGPTLSTWLDC 56 CEQDGPTLLEWLKC 57 CELVGPSLMSWLTC 58 CLTGPFVTQWLYEC59 CRAGPTLLEWLTLC 60 CADGPTLREWISFC 61 GGCTLREWLHGGFCGG 62GGCADGPTLREWISFCGG 63 GNADGPTLRQWLEGRRPKN 64 LAIEGPTLRQWLHGNGRDT 65HGRVGPTLREWKTQVATKK 66 TIKGPTLRQWLKSREHTS 67 ISDGPTLKEWLSVTRGAS 68SIEGPTLREWLTSRTPHS 69 GAREGPTLRQWLEWVRVG 70 RDLDGPTLRQWLPLPSVQ 71ALRDGPTLKQWLEYRRQA 72 ARQEGPTLKEWLFWVRMG 73 EALLGPTLREWLAWRRAQ 74MARDGPTLREWLRTYRMM 75 WMPEGPTLKQWLFHGRGQ 76 HIREGPTLRQWLVALRMV 77QLGHGPTLRQWLSWYRGM 78 ELRQGPTLHEWLQHLASK 79 VGIEGPTLRQWLAQRLNP 80WSRDGPTLREWLAWRAVG 81 AVPQGPTLKQWLLWRRCA 82 RIREGPTLKEWLAQRRGF 83RFAEGPTLREWLEQRKLV 84 DRFQGPTLREWLAAIRSV 85 AGREGPTLREWLNMRVWQ 86ALQEGPTLRQWLGWGQWG 87 YCDEGPTLKQWLVCLGLQ 88 WCKEGPTLREWLRWGFLC 89CSSGGPTLREWLQCRRMQ 90 CSWGGPTLKQWLQCVRAK 91 CQLGGPTLREWLACRLGA 92CWEGGPTLKEWLQCLVER 93 CRGGGPTLHQWLSCFRWQ 94 CRDGGPTLRQWLACLQQK 95ELRSGPTLKEWLVWRLAQ 96 GCRSGPTLREWLACREVQ 97 TCEQGPTLRQWLLCRQGR 98QGYCDEGPTLKQWLVCLGLQHS 99

TABLE 7 Ang-2 binding peptide sequences SEQUENCE SEQ ID NO. WDPWT 100WDPWTC 101 CXWDPWT (wherein X is an acidic or 102 neutral polar aminoacid residue) CXWDPWTC (wherein X is an acidic or 103 neutral polaramino acid residue) PIRQEECDWDPWTCEHMWEV 104 TNIQEECEWDPWTCDHMPGK 105WYEQDACEWDPWTCEHMAEV 106 NRLQEVCEWDPWTCEHMENV 107 AATQEECEWDPWTCEHMPRS108 LRHQEGCEWDPWTCEHMFDW 109 VPRQKDCEWDPWTCEHMYVG 110SISHEECEWDPWTCEHMQVG 111 WAAQEECEWDPWTCEHMGRM 112 TWPQDKCEWDPWTCEHMGST113 GHSQEECGWDPWTCEHMGTS 114 QHWQEECEWDPWTCDHMPSK 115NVRQEKCEWDPWTCEHMPVR 116 KSGQVECNWDPWTCEHMPRN 117 VKTQEHCDWDPWTCEHMREW118 AWGQEGCDWDPWTCEHMLPM 119 PVNQEDCEWDPWTCEHMPPM 120RAPQEDCEWDPWTCAHMDIK 121 HGQNMECEWDPWTCEHMFRY 122 PRLQEECVWDPWTCEHMPLR123 RTTQEKCEWDPWTCEHMESQ 124 QTSQEDCVWDPWTCDHMVSS 125QVIGRPCEWDPWTCEHLEGL 126 WAQQEECAWDPWTCDHMVGL 127 LPGQEDCEWDPWTCEHMVRS128 PMNQVECDWDPWTCEHMPRS 129 FGWSHGCEWDPWTCEHMGST 130KSTQDDCDWDPWTCEHMVGP 131 GPRISTCQWDPWTCEHMDQL 132 STIGDMCEWDPWTCAHMQVD133 VLGGQGCEWDPWTCRLLQGW 134 VLGGQGCQWDPWTCSHLEDG 135TTIGSMCEWDPWTCAHMQGG 136 TKGKSVCQWDPWTCSHMQSG 137 TTIGSMCQWDPWTCAHMQGG138 WVNEVVCEWDPWTCNHWDTP 139 VVQVGMCQWDPWTCKHMRLQ 140AVGSQTCEWDPWTCAHLVEV 141 QGMKMFCEWDPWTCAHIVYR 142 TTIGSMCQWDPWTCEHMQGG143 TSQRVGCEWDPWTCQHLTYT 144 QWSWPPCEWDPWTCQTVWPS 145GTSPSFCQWDPWTCSHMVQG 146 QEECEWDPWTCEHM 147 QNYKPLDELDATLYEHFIFHYT 148LNFTPLDELEQTLYEQWTLQQS 149 TKFNPLDELEQTLYEQWTLQHQ 150VKFKPLDALEQTLYEHWMFQQA 151 VKYKPLDELDEILYEQQTFQER 152TNFMPMDDLEQRLYEQFILQQG 153 SKFKPLDELEQTLYEQWTLQHA 154QKFQPLDELEQTLYEQFMLQQA 155 QNFKPMDELEDTLYKQFLFQHS 156YKFTPLDDLEQTLYEQWTLQHV 157 QEYEPLDELDETLYNQWMFHQR 158SNFMPLDELEQTLYEQFMLQHQ 159 QKYQPLDELDKTLYDQFMLQQG 160QKFQPLDELEETLYKQWTLQQR 161 VKYKPLDELDEWLYHQFTLHHQ 162QKFMPLDELDEILYEQFMFQQS 163 QTFQPLDDLEEYLYEQWIRRYH 164EDYMPLDALDAQLYEQFILLHG 165 HTFQPLDELEETLYYQWLYDQL 166YKFNPMDELEQTLYEEFLFQHA 167 TNYKPLDELDATLYEHWILQHS 168QKFKPLDELEQTLYEQWTLQQR 169 TKFQPLDELDQTLYEQWTLQQR 170TNFQPLDELDQTLYEQWTLQQR 171 KFNPLDELEETLYEQFTFQQ 172 AGGMRPYDGMLGWPNYDVQA173 QTWDDPCMHILGPVTWRRCI 174 APGQRPYDGMLGWPTYQRIV 175SGQLRPCEEIFGCGTQNLAL 176 FGDKRPLECMFGGPIQLCPR 177 GQDLRPCEDMFGCGTKDWYG178 KRPCEEIFGGCTYQ 179 GFEYCDGMEDPFTFGCDKQT 180 KLEYCDGMEDPFTQGCDNQS 181LQEWCEGVEDPFTFGCEKQR 182 AQDYCEGMEDPFTFGCEMQK 183 LLDYCEGVQDPFTFGCENLD184 HQEYCEGMEDPFTFGCEYQG 185 MLDYCEGMDDPFTFGCDKQM 186LQDYCEGVEDPFTFGCENQR 187 LQDYCEGVEDPFTFGCEKQR 188 FDYCEGVEDPFTFGCDNH 189

TABLE 8 NGF-Binding Peptide Sequences SEQUENCE SEQ ID NO.TGYTEYTEEWPMGFGYQWSF 190 TDWLSDFPFYEQYFGLMPPG 191 FMRFPNPWKLVEPPQGWYYG192 VVKAPHFEFLAPPHFHEFPF 193 FSYIWIDETPSNIDRYMLWL 194VNFPKVPEDVEPWPWSLKLY 195 TWHPKTYEEFALPFFVPEAP 196 WHFGTPYIQQQPGVYWLQAP197 VWNYGPFFMNFPDSTYFLHE 198 WRIHSKPLDYSHVWFFPADF 199FWDGNQPPDILVDWPWNPPV 200 FYSLEWLKDHSEFFQTVTEW 201 QFMELLKFFNSPGDSSHHFL202 TNVDWISNNWEHMKSFFTED 203 PNEKPYQMQSWFPPDWPVPY 204WSHTEWVPQVWWKPPNHFYV 205 WGEWINDAQVHMHEGFISES 206 VPWEHDHDLWEIISQDWHIA207 VLHLQDPRGWSNFPPGVLEL 208 IHGCWFTEEGCVWQ 209 YMQCQFARDGCPQW 210KLQCQYSESGCPTI 211 FLQCEISGGACPAP 212 KLQCEFSTSGCPDL 213 KLQCEFSTQGCPDL214 KLQCEFSTSGCPWL 215 IQGCWFTEEGCPWQ 216 SFDCDNPWGHVLQSCFGF 217SFDCDNPWGHKLQSCFGF 218

TABLE 9 Myostatin binding peptide or polypeptide sequences SEQUENCE SEQID NO: KDKCKMWHWMCKPP 616 KDLCAMWHWMCKPP 219 KDLCKMWKWMCKPP 220KDLCKMWHWMCKPK 221 WYPCYEFHFWCYDL 222 WYPCYEGHFWCYDL 223 IFGCKWWDVQCYQF224 IFGCKWWDVDCYQF 225 ADWCVSPNWFCMVM 226 HKFCPWWALFCWDF 227KDLCKMWHWMCKPP 228 IDKCAIWGWMCPPL 229 WYPCGEFGMWCLNV 230 WFTCLWNCDNE 231HTPCPWFAPLCVEW 232 KEWCWRWKWMCKPE 233 FETCPSWAYFCLDI 234 AYKCEANDWGCWWL235 NSWCEDQWHRCWWL 236 WSACYAGHFWCYDL 237 ANWCVSPNWFCMVM 238WTECYQQEFWCWNL 239 ENTCERWKWMCPPK 240 WLPCHQEGFWCMNF 241 STMCSQWHWMCNPF242 IFGCHWWDVDCYQF 243 IYGCKWWDIQCYDI 244 PDWCIDPDWWCKFW 245QGHCTRWPWMCPPY 246 WQECYREGFWCLQT 247 WFDCYGPGFKCWSP 248 GVRCPKGHLWCLYP249 HWACGYWPWSCKWV 250 GPACHSPWWWCVFG 251 TTWCISPMWFCSQQ 252HKFCPPWAIFCWDF 253 PDWCVSPRWYCNMW 254 VWKCHWFGMDCEPT 255 KKHCQIWTWMCAPK256 WFQCGSTLFWCYNL 257 WSPCYDHYFYCYTI 258 SWMCGFFKEVCMWV 259EMLCMIHPVFCNPH 260 LKTCNLWPWMCPPL 261 VVGCKWYEAWCYNK 262 PIHCTQWAWMCPPT263 DSNCPWYFLSCVIF 264 HIWCNLAMMKCVEM 265 NLQCIYFLGKCIYF 266AWRCMWFSDVCTPG 267 WFRCFLDADWCTSV 268 EKICQMWSWMCAPP 269 WFYCHLNKSECTEP270 FWRCAIGIDKCKRV 271 NLGCKWYEVWCFTY 272 IDLCNMWDGMCYPP 273EMPCNIWGWMCPPV 274 WFRCVLTGIVDWSECFGL 275 GFSCTFGLDEFYVDCSPF 276LPWCHDQVNADWGFCMLW 277 YPTCSEKFWIYGQTCVLW 278 LGPCPIHHGPWPQYCVYW 279PFPCETHQISWLGHCLSF 280 HWGCEDLMWSWHPLCRRP 281 LPLCDADMMPTIGFCVAY 282SHWCETTFWMNYAKCVHA 283 LPKCTHVPFDQGGFCLWY 284 FSSCWSPVSRQDMFCVFY 285SHKCEYSGWLQPLCYRP 286 PWWCQDNYVQHMLHCDSP 287 WFRCMLMNSFDAFQCVSY 288PDACRDQPWYMFMGCMLG 289 FLACFVEFELCFDS 290 SAYCIITESDPYVLCVPL 291PSICESYSTMWLPMCQHN 292 WLDCHDDSWAWTKMCRSH 293 YLNCVMMNTSPFVECVFN 294YPWCDGFMIQQGITCMFY 295 FDYCTWLNGFKDWKCWSR 296 LPLCNLKEISHVQACVLF 297SPECAFARWLGIEQCQRD 298 YPQCFNLHLLEWTECDWF 299 RWRCEIYDSEFLPKCWFF 300LVGCDNVWHRCKLF 301 AGWCHVWGEMFGMGCSAL 302 HHECEWMARWMSLDCVGL 303FPMCGIAGMKDFDFCVWY 304 RDDCTFWPEWLWKLCERP 305 YNFCSYLFGVSKEACQLP 306AHWCEQGPWRYGNICMAY 307 NLVCGKISAWGDEACARA 308 HNVCTIMGPSMKWFCWND 309NDLCAMWGWRNTIWCQNS 310 PPFCQNDNDMLQSLCKLL 311 WYDCNVPNELLSGLCRLF 312YGDCDQNHWMWPFTCLSL 313 GWMCHFDLHDWGATCQPD 314 YFHCMFGGHEFEVHCESF 315AYWCWHGQCVRF 316 SEHWTFTDWDGNEWWVRPF 317 MEMLDSLFELLKDMVPISKA 318SPPEEALMEWLGWQYGKFT 319 SPENLLNDLYILMTKQEWYG 320 FHWEEGIPFHVVTPYSYDRM321 KRLLEQFMNDLAELVSGHS 322 DTRDALFQEFYEFVRSRLVI 323RMSAAPRPLTYRDIMDQYWH 324 NDKAHFFEMFMFDVHNFVES 325 QTQAQKIDGLWELLQSIRNQ326 MLSEFEEFLGNLVHRQEA 327 YTPKMGSEWTSFWHNRIHYL 328 LNDTLLRELKMVLNSLSDMK329 FDVERDLMRWLEGFMQSAAT 330 HHGWNYLRKGSAPQWFEAWV 331VESLHQLQMWLDQKLASGPH 332 RATLLKDFWQLVEGYGDN 333 EELLREFYRFVSAFDY 334GLLDEFSHFIAEQFYQMPGG 335 YREMSMLEGLLDVLERLQHY 336 HNSSQMLLSELIMLVGSMMQ337 WREHFLNSDYIRDKLIAIDG 338 QFPFYVFDDLPAQLEYWIA 339EFFHWLHNHRSEVNHWLDMN 340 EALFQNFFRDVLTLSEREY 341 QYWEQQWMTYFRENGLHVQY342 NQRMMLEDLWRIMTPMFGRS 343 FLDELKAELSRHYALDDLDE 344GKLIEGLLNELMQLETFMPD 345 ILLLDEYKKDWKSWF 346QGHCTRWPWMCPPYGSGSATGGSGSTAS 347 SGSGSATGQGHCTRWPWMCPPYWYPCYEGHFWCYDLGSGSTASSGSGSAT 348 GWYPCYEGHFWCYDLHTPCPWFAPLCVEWGSGSATGGSGSTAS 349 SGSGSATGHTPCPWFAPLCVEWPDWCIDPDWWCKFWGSGSATGGSGSTA 350 SSGSGSATGPDWCIDPDWWCKFWANWCVSPNWFCMVMGSGSATGGSGSTA 351 SSGSGSATGANWCVSPNWFCMVMPDWCIDPDWWCKFWGSGSATGGSGSTA 352 SSGSGSATGPDWCIDPDWWCKFWHWACGYWPWSCKWVGSGSATGGSGST 353 ASSGSGSATGHWACGYWPWSCKWVKKHCQIWTWMCAPKGSGSATGGSGSTAS 354 SGSGSATGQGHCTRWPWMCPPYQGHCTRWPWMCPPYGSGSATGGSGSTAS 355 SGSGSATGKKHCQIWTWMCAPKKKHCQIWTWMCAPKGSGSATGGSGSTAS 356 SGSGSATGQGHCTRWPWMCPPYKKHCQIWTWMCAPKGGGGGGGGQGHC 357 TRWPWMCPPY QGHCTRWPWMCPPYGGGGGGKKHCQI 358WTWMCAPK VALHGQCTRWPWMCPPQREG 359 YPEQGLCTRWPWMCPPQTLA 360GLNQGHCTRWPWMCPPQDSN 361 MITQGQCTRWPWMCPPQPSG 362 AGAQEHCTRWPWMCAPNDWI363 GVNQGQCTRWRWMCPPNGWE 364 LADHGQCIRWPWMCPPEGWE 365ILEQAQCTRWPWMCPPQRGG 366 TQTHAQCTRWPWMCPPQWEG 367 VVTQGHCTLWPWMCPPQRWR368 IYPHDQCTRWPWMCPPQPYP 369 SYWQGQCTRWPWMCPPQWRG 370MWQQGHCTRWPWMCPPQGWG 371 EFTQWHCTRWPWMCPPQRSQ 372 LDDQWQCTRWPWMCPPQGFS373 YQTQGLCTRWPWMCPPQSQR 374 ESNQGQCTRWPWMCPPQGGW 375WTDRGPCTRWPWMCPPQANG 376 VGTQGQCTRWPWMCPPYETG 377 PYEQGKCTRWPWMCPPYEVE378 SEYQGLCTRWPWMCPPQGWK 379 TFSQGHCTRWPWMCPPQGWG 380PGAHDHCTRWPWMCPPQSRY 381 VAEEWHCRRWPWMCPPQDWR 382 VGTQGHCTRWPWMCPPQPAG383 EEDQAHCRSWPWMCPPQGWV 384 ADTQGHCTRWPWMCPPQHWF 385SGPQGHCTRWPWMCAPQGWF 386 TLVQGHCTRWPWMCPPQRWV 387 GMAHGKCTRWAWMCPPQSWK388 ELYHGQCTRWPWMCPPQSWA 389 VADHGHCTRWPWMCPPQGWG 390PESQGHCTRWPWMCPPQGWG 391 IPAHGHCTRWPWMCPPQRWR 392 FTVHGHCTRWPWMCPPYGWV393 PDFPGHCTRWRWMCPPQGWE 394 QLWQGPCTQWPWMCPPKGRY 395HANDGHCTRWQWMCPPQWGG 396 ETDHGLCTRWPWMCPPYGAR 397 GTWQGLCTRWPWMCPPQGWQ398 VATQGQCTRWPWMCPPQGWG 399 VATQGQCTRWPWMCPPQRWG 400QREWYPCYGGHLWCYDLHKA 401 ISAWYSCYAGHFWCWDLKQK 402 WTGWYQCYGGHLWCYDLRRK403 KTFWYPCYDGHFWCYNLKSS 404 ESRWYPCYEGHLWCFDLTET 405MEMLDSLFELLKDMVPISKA 406 RMEMLESLLELLKEIVPMSKAG 407RMEMLESLLELLKEIVPMSKAR 408 RMEMLESLLELLKDIVPMSKPS 409GMEMLESLFELLQEIVPMSKAP 410 RMEMLESLLELLKDIVPISNPP 411RIEMLESLLELLQEIVPISKAE 412 RMEMLQSLLELLKDIVPMSNAR 413RMEMLESLLELLKEIVPTSNGT 414 RMEMLESLFELLKEIVPMSKAG 415RMEMLGSLLELLKEIVPMSKAR 416 QMELLDSLFELLKEIVPKSQPA 417RMEMLDSLLELLKEIVPMSNAR 418 RMEMLESLLELLHEIVPMSQAG 419QMEMLESLLQLLKEIVPMSKAS 420 RMEMLDSLLELLKDMVPMTTGA 421RIEMLESLLELLKDMVPMANAS 422 RMEMLESLLQLLNEIVPMSRAR 423RMEMLESLFDLLKELVPMSKGV 424 RIEMLESLLELLKDIVPIQKAR 425RMELLESLFELLKDMVPMSDSS 426 RMEMLESLLEVLQEIVPRAKGA 427RMEMLDSLLQLLNEIVPMSHAR 428 RMEMLESLLELLKDIVPMSNAG 429RMEMLQSLFELLKGMVPISKAG 430 RMEMLESLLELLKEIVPNSTAA 431RMEMLQSLLELLKEIVPISKAG 432 RIEMLDSLLELLNELVPMSKAR 433HHGWNYLRKGSAPQWFEAWV 434 QVESLQQLLMWLDQKLASGPQG 435RMELLESLFELLKEMVPRSKAV 436 QAVSLQHLLMWLDQKLASGPQH 437DEDSLQQLLMWLDQKLASGPQL 438 PVASLQQLLIWLDQKLAQGPHA 439EVDELQQLLNWLDHKLASGPLQ 440 DVESLEQLLMWLDHQLASGPHG 441QVDSLQQVLLWLEHKLALGPQV 442 GDESLQHLLMWLEQKLALGPHG 443QIEMLESLLDLLRDMVPMSNAF 444 EVDSLQQLLMWLDQKLASGPQA 445EDESLQQLLIYLDKMLSSGPQV 446 AMDQLHQLLIWLDHKLASGPQA 447RIEMLESLLELLDEIALIPKAW 448 EVVSLQHLLMWLEHKLASGPDG 449GGESLQQLLMWLDQQLASGPQR 450 GVESLQQLLIFLDHMLVSGPHD 451NVESLEHLMMWLERLLASGPYA 452 QVDSLQQLLIWLDHQLASGPKR 453EVESLQQLLMWLEHKLAQGPQG 454 EVDSLQQLLMWLDQKLASGPHA 455EVDSLQQLLMWLDQQLASGPQK 456 GVEQLPQLLMWLEQKLASGPQR 457GEDSLQQLLMWLDQQLAAGPQV 458 ADDSLQQLLMWLDRKLASGPHV 459PVDSLQQLLIWLDQKLASGPQG 460 RATLLKDFWQLVEGYGDN 461 DWRATLLKEFWQLVEGLGDNLV462 QSRATLLKEFWQLVEGLGDKQA 463 DGRATLLTEFWQLVQGLGQKEA 464LARATLLKEFWQLVEGLGEKVV 465 GSRDTLLKEFWQLVVGLGDMQT 466DARATLLKEFWQLVDAYGDRMV 467 NDRAQLLRDFWQLVDGLGVKSW 468GVRETLLYELWYLLKGLGANQG 469 QARATLLKEFCQLVGCQGDKLS 470QERATLLKEFWQLVAGLGQNMR 471 SGRATLLKEFWQLVQGLGEYRW 472TMRATLLKEFWLFVDGQREMQW 473 GERATLLNDFWQLVDGQGDNTG 474DERETLLKEFWQLVHGWGDNVA 475 GGRATLLKELWQLLEGQGANLV 476TARATLLNELVQLVKGYGDKLV 477 GMRATLLQEFWQLVGGQGDNWM 478STRATLLNDLWQLMKGWAEDRG 479 SERATLLKELWQLVGGWGDNFG 480VGRATLLKEFWQLVEGLVGQSR 481 EIRATLLKEFWQLVDEWREQPN 482QLRATLLKEFLQLVHGLGETDS 483 TQRATLLKEFWQLIEGLGGKHV 484HYRATLLKEFWQLVDGLREQGV 485 QSRVTLLREFWQLVESYRPIVN 486LSRATLLNEFWQFVDGQRDKRM 487 WDRATLLNDFWHLMEELSQKPG 488QERATLLKEFWRMVEGLGKNRG 489 NERATLLREFWQLVGGYGVNQR 490YREMSMLEGLLDVLERLQHY 491 HQRDMSMLWELLDVLDGLRQYS 492TQRDMSMLDGLLEVLDQLRQQR 493 TSRDMSLLWELLEELDRLGHQR 494MQHDMSMLYGLVELLESLGHQI 495 WNRDMRMLESLFEVLDGLRQQV 496GYRDMSMLEGLLAVLDRLGPQL 497 TQRDMSMLEGLLEVLDRLGQQR 498WYRDMSMLEGLLEVLDRLGQQR 499 HNSSQMLLSELIMLVGSMMQ 500TQNSRQMLLSDFMMLVGSMIQG 501 MQTSRHILLSEFMMLVGSIMHG 502HDNSRQMLLSDLLHLVGTMIQG 503 MENSRQNLLRELIMLVGNMSHQ 504QDTSRHMLLREFMMLVGEMIQG 505 DQNSRQMLLSDLMILVGSMIQG 506EFFHWLHNHRSEVNHWLDMN 507 NVFFQWVQKHGRVVYQWLDINV 508FDFLQWLQNHRSEVEHWLVMDV 509

TABLE 10 BAFF binding peptide sequences SEQUENCE SEQ ID NO:PGTCFPFPWECTHA 510 WGACWPFPWECFKE 511 VPFCDLLTKHCFEA 512GSRCKYKWDVLTKQCFHH 513 LPGCKWDLLIKQWVCDPL 514 SADCYFDILTKSDVCTSS 515SDDCMYDQLTRMFICSNL 516 DLNCKYDELTYKEWCQFN 517 FHDCKYDLLTRQMVCHGL 518RNHCFWDHLLKQDICPSP 519 ANQCWWDSLTKKNVCEFF 520 YKGRQQMWDILTRSWVVSL 521QQDVGLWWDILTRAWMPNI 522 QQNAQRVWDLLIRTWVYPQ 523 GWNEAWWDELTKIWVLEQQ 524RITCDTWDSLIKKCVPQQS 525 GAIMQQFWDSLTKTWLRQS 526 WLHSGWWDPLTKHWLQQKV 527SEWFFWFDPLTRAQQLKFR 528 GVWFWWFDPLTKQWTQQAG 529 MQQCKGYYDILTKWCVTNG 530LWSKEVWDILTKSWVSQQA 531 KAAGWWFDWLTKVWVPAP 532 AYQQTWFWDSLTRLWLSTT 533SGQQHFWWDLLTRSWTPST 534 LGVGQQKWDPLTKQWVSRG 535 VGKMCQQWDPLIKRTVCVG 536CRQGAKFDLLTKQCLLGR 537 GQAIRHWDVLTKQWVDSQQ 538 RGPCGSWDLLTKHCLDSQQ 539WQWKQQQWDLLTKQMVWVG 540 PITICRKDLLTKQVVCLD 541 KTCNGKWDLLTKQCLQQQA 542KCLKGKWDLLTKQCVTEV 543 RCWNGKWDLLTKQCIHPW 544 NRDMRKWDPLIKQWIVRP 545QQAAAATWDLLTKQWLVPP 546 PEGGPKWDPLTKQQFLPPV 547 QQTPQQKKWDLLTKQWFTRN 548IGSPCKWDLLTKQMICQQT 549 CTAAGKWDLLTKQCIQQEK 550 VSQCMKWDLLTKQCLQQGW 551VWGTWKWDLLTKQYLPPQQ 552 GWWEMKWDLLTKQWYRPQQ 553 TAQQVSKWDLLTKQWLPLA 554QLWGTKWDLLTKQYIQQIM 555 WATSQKWDLLTKQWVQQNM 556 QQRQCAKWDLLTKQCVLFY 557KTTDCKWDLLTKQRICQQV 558 LLCQQGKWDLLTKQCLKLR 559 LMWFWKWDLLTKQLVPTF 560QQTWAWKWDLLTKQWIGPM 561 NKELLKWDLLTKQCRGRS 562 GQQKDLKWDLLTKQYVRQS 563PKPCQQKWDLLTKQCLGSV 564 GQIGWKWDLLTKQWIQQTR 565 VWLDWKWDLLTKQWIHPQQ 566QQEWEYKWDLLTKQWGWLR 567 HWDSWKWDLLTKQWVVQQA 568 TRPLQQKWDLLTKQWLRVG 569SDQWQQKWDLLTKQWFWDV 570 QQQTFMKWDLLTKQWIRRH 571 QQGECRKWDLLTKQCFPGQ 572GQQMGWRWDPLIKMCLGPS 573 QQLDGCKWDLLTKQKVCIP 574 HGYWQQKWDLLTKQWVSSE 575HQQGQCGWDLLTRIYLPCH 576 LHKACKWDLLTKQCWPMQQ 577 GPPGSVWDLLTKIWIQQTG 578ITQQDWRFDTLTRLWLPLR 579 QQGGFAAWDVLTKMWITVP 580 GHGTPWWDALTRIWILGV 581VWPWQQKWDLLTKQFVFQD 582 WQQWSWKWDLLTRQYISSS 583 NQQTLWKWDLLTKQFITYM 584PVYQQGWWDTLTKLYIWDG 585 WLDGGWRDPLIKRSVQQLG 586 GHQQQFKWDLLTKQWVQSN 587QQRVGQFWDVLTKMFITGS 588 QQAQGWSYDALIKTWIRWP 589 GWMHWKWDPLTKQQALPWM 590GHPTYKWDLLTKQWILQQM 591 WNNWSLWDPLTKLWLQQQN 592 WQWGWKWDLLTKQWVQQQ 593GQMGWRWDPLTKMWLGTS 594

In addition to peptides and polypeptides having amino acid sequences setforth in Tables 5-10, polypeptides that can be useful in accordance withthe invention include the Ang-2 binding polypeptide having amino acidsequence SEQ ID NO: 612:

SEQ ID NO: 612 Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro GluLeu Leu Gly Gly ProSer Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr LeuMet Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His GluAsp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn AlaLys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser ValLeu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys ValSer Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys GlyGln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu GlyGly Gln Glu Glu Cys Glu Trp Asp Pro Trp Thr Cys Glu His Met Gly Gly ThrLys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp IleAla Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr ProPro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val AspLys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu AlaLeu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys//;And the myostatin binding polypeptide having amino acid sequence SEQ IDNO: 613:

SEQ ID NO: 613 Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro GluLeu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr LeuMet Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His GluAsp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn AlaLys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser ValLeu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys ValSer Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys GlyGln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu GlyGly Leu Ala Asp His Gly Gln Cys Ile Arg Trp Pro Trp Met Cys Pro Pro GluGly Trp Glu Gly Gly Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys GlyPhe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu AsnAsn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu TyrSer Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser CysSer Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser LeuSer Pro Gly Lys//;And the EPO-mimetic polypeptide having amino acid sequence SEQ ID NO:614:

SEQ ID NO: 614 Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro GluLeu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr LeuMet Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His GluAsp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn AlaLys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser ValLeu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys ValSer Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys GlyGln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu GlyGly Gly Gly Thr Tyr Ser Cys His Phe Gly Pro Leu Thr Trp Val Cys Lys ProGln Gly Gly Gly Gly Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys GlyPhe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu AsnAsn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu TyrSer Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser CysSer Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser LeuSer Pro Gly Lys//;And the TPO-mimetic polypeptide having amino acid sequence SEQ ID NO:615:

SEQ ID NO: 615 Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro GluLeu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr LeuMet Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His GluAsp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn AlaLys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser ValLeu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys ValSer Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys GlyGln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu GlyGly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly ThrLys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp IleAla Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr ProPro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val AspLys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu AlaLeu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys//.

Pharmaceutically Acceptable Polymers.

The invention further embraces molecules covalently modified to includeone or more water soluble polymer attachments. Pharmaceuticallyacceptable polymers useful in accordance with the present inventioninclude, polyethylene glycol, polyoxyethylene glycol, or polypropyleneglycol, as described U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192; and 4,179,337. Still other useful polymers knownin the art include, but are not limited to, monomethoxy-polyethyleneglycol, dextran, cellulose, or other carbohydrate based polymers (e.g.,hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose), poly-(N-vinyl pyrrolidone)-polyethylene glycol, propyleneglycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer,polyoxyethylated polyols (e.g., glycerol) polyvinyl alcohol, polylacticacid, polyglycolic acid, copolymers of ethylene glycol and propyleneglycol, carboxymethyl cellulose, dextran, polyvinyl pyrrolidone andpolyproline, hyaluronic acid, poly-1,3-dioxolane andpoly-1,3,6-tioxocane, pectin, starch, gelatin, as well as mixtures ofany of these polymers.

A preferred polymer is polyethylene glycol (PEG). The PEG group may beof any convenient molecular weight and may be linear or branched. Theaverage molecular weight of the PEG will preferably range from about 2kiloDalton (“kD”) to about 100 kD, more preferably from about 5 kDa toabout 50 kDa, most preferably from about 5 kD to about 20 kD. The PEGgroups will generally be attached to the compounds of the invention viaacylation or reductive alkylation through a reactive group on the PEGmoiety (e.g., an aldehyde, maleimide, amino, thiol, or ester group) to areactive group on the inventive compound (e.g., an aldehyde, amino,thiol or ester group).

Covalent conjugation of proteins and peptides with poly(ethylene glycol)(PEG) has been widely recognized as an approach to significantly extendthe in vivo circulating half-lives of therapeutic proteins. PEGylationachieves this effect predominately by retarding renal clearance, sincethe PEG moiety adds considerable hydrodynamic radius to the protein.(Zalipsky, S., et al., Use of functionalized poly(ethylene glycol)s formodification of polypeptides., in poly(ethylene glycol) chemistry:Biotechnical and biomedical applications., J. M. Harris, Ed., PlenumPress: New York., 347-370 (1992)). Additional benefits often conferredby PEGylation of proteins and peptides include increased solubility,resistance to proteolytic degradation, and reduced immunogenicity of thetherapeutic polypeptide. The merits of protein PEGylation are evidencedby the commercialization of several PEGylated proteins includingPEG-Adenosine deaminase (Adagen™/Enzon Corp.), PEG-L-asparaginase(Oncaspar™/Enzon Corp.), PEG-Interferon α-2b(PEG-Intron™/Schering/Enzon), PEG-Interferon α-2a (PEGASYS™/Roche) andPEG-G-CSF (Neulasta™/Amgen) as well as many others in clinical trials.

Briefly, the PEG groups are generally attached to the peptide portion ofthe composition of the invention via acylation or reductive alkylationthrough a reactive group on the PEG moiety (e.g., an aldehyde, amino,thiol, or ester group) to a reactive group on the inventive compound(e.g., an aldehyde, amino, or ester group).

A useful strategy for the PEGylation of synthetic peptides consists ofcombining, through forming a conjugate linkage in solution, apolypeptide or peptide and a PEG moiety, each bearing a specialfunctionality that is mutually reactive toward the other. Thepolypeptides or peptides can be easily prepared with conventional solidphase synthesis. The polypeptides or peptides are “preactivated” with anappropriate functional group at a specific site. The precursors arepurified and fully characterized prior to reacting with the PEG moiety.Ligation of the polypeptide or peptide with PEG usually takes place inaqueous phase and can be easily monitored by reverse phase analyticalHPLC. The PEGylated polypeptides or peptides can be easily purified bypreparative HPLC and characterized by analytical HPLC, amino acidanalysis and laser desorption mass spectrometry.

PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods well known in the art (Sandler and Karo,Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). Inthe present application, the term “PEG” is used broadly to encompass anypolyethylene glycol molecule, in mono-, bi-, or poly-functional form,without regard to size or to modification at an end of the PEG, and canbe represented by the formula:X—O(CH₂CH₂O)_(n-1)CH₂CH₂OH,  (XXIX)

where n is 20 to 2300 and X is H or a terminal modification, e.g., aC₁₋₄ alkyl.

In some useful embodiments, a PEG used in the invention terminates onone end with hydroxy or methoxy, i.e., X is H or CH₃ (“methoxy PEG”). Itis noted that the other end of the PEG, which is shown in formula (II)terminating in OH, covalently attaches to an activating moiety via anether oxygen bond, an amine linkage, or amide linkage. When used in achemical structure, the term “PEG” includes the formula (II) abovewithout the hydrogen of the hydroxyl group shown, leaving the oxygenavailable to react with a free carbon atom of a linker to form an etherbond. More specifically, in order to conjugate PEG to a peptide, thepeptide must be reacted with PEG in an “activated” form. Activated PEGcan be represented by the formula:(PEG)-(A)  (XXX)where PEG (defined supra) covalently attaches to a carbon atom of theactivation moiety (A) to form an ether bond, an amine linkage, or amidelinkage, and (A) contains a reactive group which can react with anamino, imino, or thiol group on an amino acid residue of a polypeptide,peptide or a linker moiety covalently attached to the peptide portion.

Techniques for the preparation of activated PEG and its conjugation tobiologically active peptides are well known in the art. (E.g., see U.S.Pat. Nos. 5,643,575, 5,919,455, 5,932,462, and 5,990,237; Thompson etal., PEGylation of polypeptides, EP 0575545 B1; Petit, Site specificprotein modification, U.S. Pat. Nos. 6,451,986, and 6,548,644; S. Hermanet al., Poly(ethylene glycol) with reactive endgroups: I. Modificationof proteins, J. Bioactive Compatible Polymers, 10:145-187 (1995); Y. Luet al., Pegylated peptides III: Solid-phase synthesis with PEGylatingreagents of varying molecular weight: synthesis of multiply PEGylatedpeptides, Reactive Polymers, 22:221-229 (1994); A. M. Felix et al.,PEGylated Peptides IV: Enhanced biological activity of site-directedPEGylated GRF analogs, Int. J. Peptide Protein Res., 46:253-264 (1995);A. M. Felix, Site-specific poly(ethylene glycol)ylation of peptides, ACSSymposium Series 680(poly(ethylene glycol)): 218-238 (1997); Y. Ikeda etal., Polyethylene glycol derivatives, their modified peptides, methodsfor producing them and use of the modified peptides, EP 0473084 B1; G.E. Means et al., Selected techniques for the modification of proteinside chains, in: Chemical modification of proteins, Holden Day, Inc.,219 (1971)).

Activated PEG, such as PEG-aldehydes or PEG-aldehyde hydrates, can bechemically synthesized by known means or obtained from commercialsources, e.g., Shearwater Polymers, (Huntsville, Ala.) or Enzon, Inc.(Piscataway, N.J.).

An example of a useful activated PEG for purposes of the presentinvention is a PEG-aldehyde compound (e.g., a methoxy PEG-aldehyde),such as PEG-propionaldehyde, which is commercially available fromShearwater Polymers (Huntsville, Al). PEG-propionaldehyde is representedby the formula PEG-CH₂CH₂CHO. (See, e.g., U.S. Pat. No. 5,252,714).Other examples of useful activated PEG are PEG acetaldehyde hydrate andPEG bis aldehyde hydrate, which latter yields a bifunctionally activatedstructure. (See, e.g., Bentley et al., Poly(ethylene glycol) aldehydehydrates and related polymers and applications in modifying amines, U.S.Pat. No. 5,990,237).

Another useful activated PEG for generating PEG-conjugated polypeptidesor peptides of the present invention is a PEG-maleimide compound, suchas, but not limited to, a methoxy PEG-maleimide, such as maleimidomonomethoxy PEG, are particularly useful for generating thePEG-conjugated peptides of the invention. (E.g., Shen, N-maleimidylpolymer derivatives, U.S. Pat. No. 6,602,498; C. Delgado et al., Theuses and properties of PEG-linked proteins., Crit. Rev. Therap. DrugCarrier Systems, 9:249-304 (1992); S. Zalipsky et al., Use offunctionalized poly(ethylene glycol)s for modification of polypeptides,in: Poly(ethylene glycol) chemistry: Biotechnical and biomedicalapplications (J. M. Harris, Editor, Plenum Press: New York, 347-370(1992); S. Herman et al., Poly(ethylene glycol) with reactive endgroups:I. Modification of proteins, J. Bioactive Compatible Polymers,10:145-187 (1995); P. J. Shadle et al., Conjugation of polymer to colonystimulating factor-1, U.S. Pat. No. 4,847,325; G. Shaw et al., Cysteineadded variants IL-3 and chemical modifications thereof, U.S. Pat. No.5,166,322 and EP 0469074 B1; G. Shaw et al., Cysteine added variants ofEPO and chemical modifications thereof, EP 0668353 A1; G. Shaw et al.,Cysteine added variants G-CSF and chemical modifications thereof, EP0668354 A1; N. V. Katre et al., Interleukin-2 muteins and polymerconjugation thereof, U.S. Pat. No. 5,206,344; R. J. Goodson and N. V.Katre, Site-directed pegylation of recombinant interleukin-2 at itsglycosylation site, Biotechnology, 8:343-346 (1990)).

A poly(ethylene glycol) vinyl sulfone is another useful activated PEGfor generating the PEG-conjugated peptides of the present invention byconjugation at thiolated amino acid residues, e.g., at C residues.(E.g., M. Morpurgo et al., Preparation and characterization ofpoly(ethylene glycol) vinyl sulfone, Bioconj. Chem., 7:363-368 (1996);see also Harris, Functionalization of polyethylene glycol for formationof active sulfone-terminated PEG derivatives for binding to proteins andbiologically compatible materials, U.S. Pat. Nos. 5,446,090; 5,739,208;5,900,461; 6,610,281 and 6,894,025; and Harris, Water soluble activesulfones of poly(ethylene glycol), WO 95/13312 A1).

Another activated form of PEG that is useful in accordance with thepresent invention, is a PEG-N-hydroxysuccinimide ester compound, forexample, methoxy PEG-N-hydroxysuccinimidyl (NHS) ester.

Heterobifunctionally activated forms of PEG are also useful. (See, e.g.,Thompson et al., PEGylation reagents and biologically active compoundsformed therewith, U.S. Pat. No. 6,552,170).

Typically, a polypeptide or peptide of interest is reacted by knownchemical techniques with an activated PEG compound, such as but notlimited to, a thiol-activated PEG compound, a diol-activated PEGcompound, a PEG-hydrazide compound, a PEG-oxyamine compound, or aPEG-bromoacetyl compound. (See, e.g., S. Herman, Poly(ethylene glycol)with Reactive Endgroups: I. Modification of Proteins, J. Bioactive andCompatible Polymers, 10:145-187 (1995); S. Zalipsky, Chemistry ofPolyethylene Glycol Conjugates with Biologically Active Molecules,Advanced Drug Delivery Reviews, 16:157-182 (1995); R. Greenwald et al.,Poly(ethylene glycol) conjugated drugs and prodrugs: a comprehensivereview, Critical Reviews in Therapeutic Drug Carrier Systems, 17:101-161(2000)).

Polysaccharide polymers are another type of water soluble polymer whichmay be used for protein modification. Dextrans are polysaccharidepolymers comprised of individual subunits of glucose predominantlylinked by α1-6 linkages. The dextran itself is available in manymolecular weight ranges, and is readily available in molecular weightsfrom about 1 kD to about 70 kD. Dextran is a suitable water solublepolymer for use in the present invention by itself or in combinationwith a different additional functional moiety (e.g., Fc). See, forexample, WO 96/11953 and WO 96/05309. The use of dextran conjugated totherapeutic or diagnostic immunoglobulins has been reported; see, forexample, European Patent Publication No. 0 315 456, which is herebyincorporated by reference. Dextran of about 1 kD to about 20 kD ispreferred when dextran is used in accordance with the present invention.

Linkers.

Any “linker” group is optional. When present, its chemical structure isnot critical, since it serves primarily as a spacer, which can be usefulin optimizing pharamcologial activity of some embodiments of theinventive composition. The linker is preferably made up of amino acidslinked together by peptide bonds. As stated herein above, the linkermoiety, if present, can be independently the same or different from anyother linker, or linkers, that may be present in the inventivecomposition. For example, an “(L)_(c)” can represent the same linkermoiety as, or a different linker moiety from, any other “(L)_(c)” or any“(L)_(d)”, “(L)_(e)”, or “(L)_(f)”, in accordance with the invention.The linker is preferably made up of amino acids linked together bypeptide bonds. Thus, in some embodiments, the linker is made up of from1 to about 30 amino acids linked by peptide bonds, wherein the aminoacids are selected from the 20 naturally occurring amino acids. Some ofthese amino acids may be glycosylated, as is well understood by those inthe art. For example, a useful linker sequence constituting asialylation site is X₁X₂NX₄X₅G (SEQ ID NO: 619), wherein X₁, X₂, X₄ andX₅ are each independently any amino acid residue.

In a more preferred embodiment, the 1 to 30 amino acids are selectedfrom glycine, alanine, proline, asparagine, glutamine, and lysine. Evenmore preferably, a linker is made up of a majority of amino acids thatare sterically unhindered, such as glycine and alanine. Thus, preferredlinkers include polyglycines (particularly (Gly)4, (Gly)5),poly(Gly-Ala), and polyalanines. Other preferred linkers are thoseidentified herein as “L5” (GGGGS; SEQ ID NO: 620), “L10” (GGGGSGGGGS;SEQ ID NO: 621), “L25” GGGGSGGGGSGGGGSGGGGSGGGGS; SEQ ID NO: 622) andany linkers used in the working examples hereinafter. The linkersdescribed herein, however, are exemplary; linkers within the scope ofthis invention can be much longer and can include other residues. Thus,preferred linkers are polyglycines (particularly (Gly)₄, (Gly)₅),poly(Gly-Ala), and polyalanines. Other specific examples of linkers are:

(SEQ ID NO: 595) (Gly)₃Lys(Gly)₄; (SEQ ID NO: 596)(Gly)₃AsnGlySer(Gly)₂; (SEQ ID NO: 597) (Gly)₃Cys(Gly)₄; and (SEQ ID NO:598) GlyProAsnGlyGly.To explain the above nomenclature, for example, (Gly)₃Lys(Gly)₄ meansGly-Gly-Gly-Lys-Gly-Gly-Gly-Gly. Combinations of Gly and Ala are alsopreferred. The linkers shown here are exemplary; linkers within thescope of this invention may be much longer and may include otherresidues.

In some embodiments of the compositions of this invention, whichcomprise a peptide linker moiety (L), acidic residues, for example,glutamate or aspartate residues, are placed in the amino acid sequenceof the linker moiety (L). Examples include the following peptide linkersequences:

(SEQ ID NO: 623) GGEGGG; (SEQ ID NO: 624) GGEEEGGG; (SEQ ID NO: 625)GEEEG; (SEQ ID NO: 626) GEEE; (SEQ ID NO: 627) GGDGGG; (SEQ ID NO: 628)GGDDDGG; (SEQ ID NO: 629) GDDDG; (SEQ ID NO: 630) GDDD; (SEQ ID NO: 631)GGGGSDDSDEGSDGEDGGGGS; (SEQ ID NO: 632) WEWEW; (SEQ ID NO: 633) FEFEF;(SEQ ID NO: 634) EEEWWW; (SEQ ID NO: 635) EEEFFF; (SEQ ID NO: 636)WWEEEWW; or (SEQ ID NO: 637) FFEEEFF.

In other embodiments, the linker constitutes a phosphorylation site,e.g., X₁X₂YX₃X₄G (SEQ ID NO: 638), wherein X₁, X₂, X₃ and X₄ are eachindependently any amino acid residue; X₁X₂SX₃X₄G (SEQ ID NO: 639),wherein X₁, X₂, X₃ and X₄ are each independently any amino acid residue;or X₁X₂TX₃X₄G (SEQ ID NO: 640), wherein X₁, X₂, X₃ and X₄ are eachindependently any amino acid residue.

Non-peptide linkers are also possible. For example, alkyl linkers suchas —NH—(CH₂)_(s)—C(O)—, wherein s=2-20 could be used. These alkyllinkers may further be substituted by any non-sterically hindering groupsuch as lower alkyl (e.g., C₁-C₆) lower acyl, halogen (e.g., Cl, Br),CN, NH₂, phenyl, etc. An exemplary non-peptide linker is a PEG linker,

wherein n is such that the linker has a molecular weight of 100 to 5000kD, preferably 100 to 500 kD. The peptide linkers may be altered to formderivatives in the same manner as described above.

Derivatives.

The compositions of matter of the present invention also encompass“derivatives” that include polypeptide or peptide portions bearingmodifications other than, or in addition to, insertions, deletions, orsubstitutions of amino acid residues. Preferably, the modifications arecovalent in nature, and include for example, chemical bonding withpolymers, lipids, other organic, and inorganic moieties. Derivatives ofthe invention may be prepared to increase circulating half-life of amolecule; to improve targeting capacity for the molecule to desiredcells, tissues, or organs; to improve the solubility or absorption of amolecule; or to eliminate or attenuate any undesirable side-effect of amolecule.

Exemplary derivatives include compounds in which:

-   -   1. The compound or some portion thereof is cyclic. For example,        the peptide portion may be modified to contain two or more Cys        residues (e.g., in the linker), which could cyclize by disulfide        bond formation.    -   2. The compound is cross-linked or is rendered capable of        cross-linking between molecules. For example, the peptide        portion may be modified to contain one Cys residue and thereby        be able to form an intermolecular disulfide bond with a like        molecule. The compound may also be cross-linked through its        C-terminus, as in the molecule shown below.

-   -   3. One or more peptidyl [—C(O)NR-] linkages (bonds) is replaced        by a non-peptidyl linkage. Exemplary non-peptidyl linkages are        —CH₂-carbamate [—CH₂—OC(O)NR—]phosphonate, —CH₂-sulfonamide        [—CH₂—S(O)₂NR-], urea [—NHC(O)NH-], —CH₂-secondary amine, and        alkylated peptide [—C(O)NR⁶— wherein R⁶ is lower alkyl].    -   4. The N-terminus is derivatized. Typically, the N-terminus may        be acylated or modified to a substituted amine Exemplary        N-terminal derivative groups include —NRR¹ (other than —NH₂),        —NRC(O)R¹, —NRC(O)OR¹, —NRS(O)₂R¹, —NHC(O)NHR¹, succinimide, or        benzyloxycarbonyl-NH—(CBZ—NH—), wherein R and R¹ are each        independently hydrogen or lower alkyl and wherein the phenyl        ring may be substituted with 1 to 3 substituents selected from        the group consisting of C₁-C₄ alkyl, C₁-C₄ alkoxy, chloro, and        bromo.    -   5. The free C-terminus is derivatized. Typically, the C-terminus        is esterified or amidated. For example, one may use methods        described in the art to add (NH—CH₂—CH₂—NH₂)₂ to compounds of        this invention. Likewise, one may use methods described in the        art to add —NH₂ to compounds of this invention. Exemplary        C-terminal derivative groups include, for example, —C(O)R²        wherein R² is lower alkoxy or —NR³R⁴ wherein R³ and R⁴ are        independently hydrogen or C₁-C₈ alkyl (preferably C₁-C₄ alkyl).    -   6. A disulfide bond is replaced with another, preferably more        stable, cross-linking moiety (e.g., an alkylene). See, e.g.,        Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9; Alberts et        al. (1993) Thirteenth Am. Pep. Symp., 357-9.    -   7. One or more individual amino acid residues is modified.        Various derivatizing agents are known to react specifically with        selected side chains or terminal residues, as described in        detail below.

Lysinyl residues and amino terminal residues may be reacted withsuccinic or other carboxylic acid anhydrides, which reverse the chargeof the lysinyl residues. Other suitable reagents for derivatizingalpha-amino-containing residues include imidoesters such as methylpicolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride;trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; andtransaminase-catalyzed reaction with glyoxylate.

Arginyl residues may be modified by reaction with any one or combinationof several conventional reagents, including phenylglyoxal,2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization ofarginyl residues requires that the reaction be performed in alkalineconditions because of the high pKa of the guanidine functional group.Furthermore, these reagents may react with the groups of lysine as wellas the arginine epsilon-amino group.

Specific modification of tyrosyl residues has been studied extensively,with particular interest in introducing spectral labels into tyrosylresidues by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidizole andtetranitromethane are used to form O-acetyl tyrosyl species and 3-nitroderivatives, respectively.

Carboxyl side chain groups (aspartyl or glutamyl) may be selectivelymodified by reaction with carbodiimides (R′—N═C═N—R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues may be converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues may be deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Cysteinyl residues can be replaced by amino acid residues or othermoieties either to eliminate disulfide bonding or, conversely, tostabilize cross-linking. See, e.g., Bhatnagar et al. (1996), J. Med.Chem. 39: 3814-9.

Derivatization with bifunctional agents is useful for cross-linking thepeptides or their functional derivatives to a water-insoluble supportmatrix or to other macromolecular vehicles. Commonly used cross-linkingagents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),and bifunctional maleimides such as bis-N-maleimido-1,8-octane.Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Carbohydrate (oligosaccharide) groups may conveniently be attached tosites that are known to be glycosylation sites in proteins. Generally,O-linked oligosaccharides are attached to serine (Ser) or threonine(Thr) residues while N-linked oligosaccharides are attached toasparagine (Asn) residues when they are part of the sequenceAsn-X-Ser/Thr, where X can be any amino acid except proline. X ispreferably one of the 19 naturally occurring amino acids other thanproline. The structures of N-linked and O-linked oligosaccharides andthe sugar residues found in each type are different. One type of sugarthat is commonly found on both is N-acetylneuraminic acid (referred toas sialic acid). Sialic acid is usually the terminal residue of bothN-linked and O-linked oligosaccharides and, by virtue of its negativecharge, may confer acidic properties to the glycosylated compound. Suchsite(s) may be incorporated in the linker of the compounds of thisinvention and are preferably glycosylated by a cell during recombinantproduction of the polypeptide compounds (e.g., in mammalian cells suchas CHO, BHK, COS). However, such sites may further be glycosylated bysynthetic or semi-synthetic procedures known in the art.

Other possible modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, oxidation of the sulfur atom in Cys, methylation of thealpha-amino groups of lysine, arginine, and histidine side chains.Creighton, Proteins: Structure and Molecule Properties (W. H. Freeman &Co., San Francisco), pp. 79-86 (1983).

Such derivatized moieties preferably improve one or more characteristicsincluding anti-angiogenic activity, solubility, absorption, biologicalhalf life, and the like of the compounds. Alternatively, derivatizedmoieties may result in compounds that have the same, or essentially thesame, characteristics and/or properties of the compound that is notderivatized. The moieties may alternatively eliminate or attenuate anyundesirable side effect of the compounds and the like.

Compounds of the present invention may be changed at the DNA level, aswell. The DNA sequence encoding any portion of the compound may bechanged to codons more compatible with the chosen host cell. For E.coli, which is the preferred host cell, optimized codons are known inthe art. Codons may be substituted to eliminate restriction sites or toinclude silent restriction sites, which may aid in processing of the DNAin the selected host cell. The vehicle, linker and peptide DNA sequencesmay be modified to include any of the foregoing sequence changes.

Isotope- and Toxin-Conjugated Derivatives.

Another set of useful derivatives are the above-described moleculesconjugated to toxins, tracers, or radioisotopes. Such conjugation isespecially useful for molecules comprising peptide sequences that bindto tumor cells or pathogens. Such molecules may be used as therapeuticagents or as an aid to surgery (e.g., radioimmunoguided surgery or RIGS)or as diagnostic agents (e.g., radioimmunodiagnostics or RID).

As therapeutic agents, these conjugated derivatives possess a number ofadvantages. They facilitate use of toxins and radioisotopes that wouldbe toxic if administered without the specific binding provided by thepeptide sequence. They also can reduce the side-effects that attend theuse of radiation and chemotherapy by facilitating lower effective dosesof the conjugation partner.

Useful conjugation partners include:

-   -   radioisotopes, such as ⁹⁰Yttrium, ¹³¹Iodine, ²²⁵Actinium, and        ²¹³Bismuth;    -   ricin A toxin, microbially derived toxins such as Pseudomonas        endotoxin (e.g., PE38, PE40), and the like;    -   partner molecules in capture systems (see below);    -   biotin, streptavidin (useful as either partner molecules in        capture systems or as tracers, especially for diagnostic use);        and    -   cytotoxic agents (e.g., doxorubicin).

One useful adaptation of these conjugated derivatives is use in acapture system. In such a system, the molecule of the present inventionwould comprise a benign capture molecule. This capture molecule would beable to specifically bind to a separate effector molecule comprising,for example, a toxin or radioisotope. Both the vehicle-conjugatedmolecule and the effector molecule would be administered to the patient.In such a system, the effector molecule would have a short half-lifeexcept when bound to the vehicle-conjugated capture molecule, thusminimizing any toxic side-effects. The vehicle-conjugated molecule wouldhave a relatively long half-life but would be benign and non-toxic. Thespecific binding portions of both molecules can be part of a knownspecific binding pair (e.g., biotin, streptavidin) or can result frompeptide generation methods such as those described herein.

Such conjugated derivatives may be prepared by methods known in the art.In the case of protein effector molecules (e.g., Pseudomonas endotoxin),such molecules can be expressed as fusion proteins from correlative DNAconstructs. Radioisotope conjugated derivatives may be prepared, forexample, as described for the BEXA antibody (Coulter). Derivativescomprising cytotoxic agents or microbial toxins may be prepared, forexample, as described for the BR96 antibody (Bristol-Myers Squibb).Molecules employed in capture systems may be prepared, for example, asdescribed by the patents, patent applications, and publications fromNeoRx. Molecules employed for RIGS and RID may be prepared, for example,by the patents, patent applications, and publications from NeoProbe.

Preparing a peptide derivative for conjugation to a Fc domain inaccordance with the present invention can be useful. Tumor cells, forexample, exhibit epitopes not found on their normal counterparts. Suchepitopes include, for example, different post-translationalmodifications resulting from their rapid proliferation. Thus, one aspectof this invention is a process comprising:

-   -   a) selecting at least one randomized peptide that specifically        binds to a target epitope; and    -   b) preparing a pharmacologic agent comprising (i) at least one        Fc domain monomer, (ii) at least one amino acid sequence of the        selected peptide or peptides, and (iii) an effector molecule.        The target epitope is preferably a tumor-specific epitope or an        epitope specific to a pathogenic organism. The effector molecule        may be any of the above-noted conjugation partners and is        preferably a radioisotope.

Variants.

Variants of polypeptide or peptide portions of the inventive compositionof matter (e.g., additional functional moiety, linker, or Fc domainportions), are also included within the scope of the present invention.Included within variants are insertional, deletional, and substitutionalvariants. It is understood that a particular molecule of the presentinvention may contain one, two or all three types of variantpolypeptides or peptides. Insertional and substitutional variants maycontain canonical amino acids, non-canonical amino acids (as set forthherein), or both. It is also understood that, in accordance with thepresent invention, polypeptide or peptide variants can be made beforechemical conjugation to an Fc domain or can be designed to be expressedas part of a fusion protein with the Fc domain, as desired in variousembodiments of the inventive composition of matter.

In one example, insertional variants are provided wherein one or moreamino acid residues, either naturally occurring or unconventional aminoacids, supplement a peptide or a polypeptide amino acid sequence.Insertions may be located at either or both termini, or may bepositioned within internal regions of the amino acid sequence.Insertional variants with additional residues at either or both terminican include, for example, fusion proteins and proteins including aminoacid tags or labels. Insertional variants include peptides andpeptibodies wherein one or more amino acid residues are added to thepeptide or polypeptide amino acid sequence, or fragment thereof.

Variants of the invention also include mature peptides and polypeptideswherein leader or signal sequences are removed, and the resultingproteins having additional amino terminal residues, which amino acidsmay be natural or non-natural. Molecules of this invention (such aspeptibodies) with an additional methionyl residue at amino acid position−1 (Met⁻¹-peptibody) are contemplated, as are specific binding agentswith additional methionine and lysine residues at positions −2 and −1(Met⁻²-Lys⁻¹-) conjugated to Fc domain as additional moieties inaccordance with the invention. Variants having additional Met, Met-Lys,Lys residues (or one or more basic residues, in general) areparticularly useful for enhanced recombinant protein production inbacterial host cells.

The invention also embraces variants having additional amino acidresidues that arise from use of specific expression systems. Forexample, use of commercially available vectors that express a desiredpolypeptide as part of glutathione-S-transferase (GST) fusion productprovides the desired polypeptide having an additional glycine residue atamino acid position −1 after cleavage of the GST component from thedesired polypeptide. Variants which result from expression in othervector systems are also contemplated, including those whereinpoly-histidine tags are incorporated into the amino acid sequence,generally at the carboxy and/or amino terminus of the sequence.

Insertional variants also include fusion proteins wherein the aminoand/or carboxy termini of the peptide or peptibody is fused to anotherpolypeptide, a fragment thereof or amino acids which are not generallyrecognized to be part of any specific protein sequence. Examples of suchfusion proteins are immunogenic polypeptides, proteins with longcirculating half lives, such as immunoglobulin constant regions, markerproteins, proteins or polypeptides that facilitate purification of thedesired peptide or peptibody, and polypeptide sequences that promoteformation of multimeric proteins (such as leucine zipper motifs that areuseful in dimer formation/stability).

This type of insertional variant generally has all or a substantialportion of the native molecule, linked at the N- or C-terminus, to allor a portion of a second polypeptide. For example, fusion proteinstypically employ leader sequences from other species to permit therecombinant expression of a protein in a heterologous host. Anotheruseful fusion protein includes the addition of an immunologically activedomain, such as an antibody epitope, to facilitate purification of thefusion protein. Inclusion of a cleavage site at or near the fusionjunction will facilitate removal of the extraneous polypeptide afterpurification. Other useful fusions include linking of functionaldomains, such as active sites from enzymes, glycosylation domains,cellular targeting signals or transmembrane regions.

There are various commercially available fusion protein expressionsystems that may be used in the present invention. Particularly usefulsystems include but are not limited to the glutathione-S-transferase(GST) system (Pharmacia), the maltose binding protein system (NEB,Beverley, Mass.), the FLAG system (IBI, New Haven, Conn.), and the 6×Hissystem (Qiagen, Chatsworth, Calif.). These systems are capable ofproducing recombinant peptides and/or peptibodies bearing only a smallnumber of additional amino acids, which are unlikely to significantlyaffect the activity of the peptide or peptibody. For example, both theFLAG system and the 6×His system add only short sequences, both of whichare known to be poorly antigenic and which do not adversely affectfolding of a polypeptide to its native conformation. Another N-terminalfusion that is contemplated to be useful is the fusion of a Met-Lysdipeptide at the N-terminal region of the protein or peptides. Such afusion may produce beneficial increases in protein expression oractivity.

Other fusion systems produce polypeptide hybrids where it is desirableto excise the fusion partner from the desired peptide or peptibody. Inone embodiment, the fusion partner is linked to the recombinantpeptibody by a peptide sequence containing a specific recognitionsequence for a protease. Examples of suitable sequences are thoserecognized by the Tobacco Etch Virus protease (Life Technologies,Gaithersburg, Md.) or Factor Xa (New England Biolabs, Beverley, Mass.).

In some embodiments of the inventive composition of matter, fusionpolypeptides comprise all or part. of the molecule, in combination withtruncated tissue factor (tTF). tTF is a vascular targeting agentconsisting of a truncated form of a human coagulation-inducing proteinthat acts as a tumor blood vessel clotting agent, as described U.S. Pat.Nos. 5,877,289; 6,004,555; 6,132,729; 6,132,730; 6,156,321; and EuropeanPatent No. EP 0988056. The fusion of tTF to the anti-Ang-2 peptibody orpeptide, or fragments thereof facilitates the delivery of anti-Ang-2 totarget cells.

In some embodiments of the present invention, deletion variants can beuseful, wherein one or more amino acid residues in a peptide orpolypeptide portion of the composition of matter are removed. Deletionscan be effected at one or both termini of the polypeptide or peptideportion, or from removal of one or more non-terminal residues within theamino acid sequence. Deletion variants necessarily include all fragmentsof a peptide or polypeptide portion of the inventive composition ofmatter.

In other embodiments of the present invention, substitution variants canbe useful. Substitution variants include those peptides and polypeptideportions wherein one or more amino acid residues are removed andreplaced with one or more alternative amino acids, which amino acids maybe naturally occurring or non-naturally occurring. Substitutionalvariants generate peptides or polypeptides that are “similar” to theoriginal peptide or polypeptide, in that the two have sequences with acertain percentage of amino acids that are identical. Substitutionvariants include substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, amino acids within a peptide or peptibody, wherein the number ofsubstitutions may be up to ten percent or more, of the amino acids ofthe peptide or peptibody. The substitutions can be conservative innature, however, the invention embraces substitutions that are alsonon-conservative and also includes non-canonical amino acids.

Identity and similarity of related peptides and peptibodies can bereadily calculated by known methods. Such methods include, but are notlimited to, those described in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York (1988); Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork (1993); Computer Analysis of Sequence Data, Part 1, Griffin, A. M.,and Griffin, H. G., eds., Humana Press, New Jersey (1994); SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press (1987);Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M.Stockton Press, New York (1991); and Carillo et al., SIAM J. AppliedMath., 48:1073 (1988).

Preferred methods to determine the relatedness or percent identity oftwo peptides or polypeptides, or a polypeptide and a peptide, aredesigned to give the largest match between the sequences tested. Methodsto determine identity are described in publicly available computerprograms. Preferred computer program methods to determine identitybetween two sequences include, but are not limited to, the GCG programpackage, including GAP (Devereux et al., Nucl. Acid. Res., 12:387(1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.,BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol., 215:403-410(1990)). The BLASTX program is publicly available from the NationalCenter for Biotechnology Information (NCBI) and other sources (BLASTManual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul etal., supra (1990)). The well-known Smith Waterman algorithm may also beused to determine identity.

Certain alignment schemes for aligning two amino acid sequences mayresult in the matching of only a short region of the two sequences, andthis small aligned region may have very high sequence identity eventhough there is no significant relationship between the two full-lengthsequences. Accordingly, in certain embodiments, the selected alignmentmethod (GAP program) will result in an alignment that spans at least tenpercent of the full length of the target polypeptide being compared,i.e., at least 40 contiguous amino acids where sequences of at least 400amino acids are being compared, 30 contiguous amino acids wheresequences of at least 300 to about 400 amino acids are being compared,at least 20 contiguous amino acids where sequences of 200 to about 300amino acids are being compared, and at least 10 contiguous amino acidswhere sequences of about 100 to 200 amino acids are being compared.

For example, using the computer algorithm GAP (Genetics Computer Group,University of Wisconsin, Madison, Wis.), two polypeptides for which thepercent sequence identity is to be determined are aligned for optimalmatching of their respective amino acids (the “matched span”, asdetermined by the algorithm). In certain embodiments, a gap openingpenalty (which is typically calculated as 3× the average diagonal; the“average diagonal” is the average of the diagonal of the comparisonmatrix being used; the “diagonal” is the score or number assigned toeach perfect amino acid match by the particular comparison matrix) and agap extension penalty (which is usually 1/10 times the gap openingpenalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62are used in conjunction with the algorithm. In certain embodiments, onemay also use a standard comparison matrix in the algorithm, See Dayhoffet al., Atlas of Protein Sequence and Structure, 5(3)(1978) for the PAM250 comparison matrix; and Henikoff et al., Proc. Natl. Acad. Sci USA,89:10915-10919 (1992) for the BLOSUM 62 comparison matrix.

In certain embodiments, the parameters for a polypeptide sequencecomparison include the following:

Algorithm: Needleman et al., J. Mol. Biol., 48:443-453 (1970);

Comparison matrix: BLOSUM 62 from Henikoff et al., supra (1992);

Gap Penalty: 12

Gap Length Penalty: 4

Threshold of Similarity: 0

The GAP program may be useful with the above parameters. In certainembodiments, the aforementioned parameters are the default parametersfor polypeptide comparisons (along with no penalty for end gaps) usingthe GAP algorithm.

In certain embodiments, the parameters for polynucleotide moleculesequence (as opposed to an amino acid sequence) comparisons include thefollowing:

Algorithm: Needleman et al., supra (1970);

Comparison matrix: matches=+10, mismatch=0

Gap Penalty: 50

Gap Length Penalty: 3

The GAP program may also be useful with the above parameters. Theaforementioned parameters are the default parameters for polynucleotidemolecule comparisons.

Other exemplary algorithms, gap opening penalties, gap extensionpenalties, comparison matrices, thresholds of similarity, etc. may beused, including those set forth in the Program Manual, WisconsinPackage, Version 9, September, 1997. The particular choices to be madewill be apparent to those of skill in the art and will depend on thespecific comparison to be made, such as DNA-to-DNA, protein-to-protein,protein-to-DNA; and additionally, whether the comparison is betweengiven pairs of sequences (in which case GAP or BestFit are generallypreferred) or between one sequence and a large database of sequences (inwhich case FASTA or BLASTA are preferred).

It will be appreciated that amino acid residues can be divided intoclasses based on their common side chain properties:

-   -   1. Neutral Hydrophobic: Alanine (Ala; A), Valine (Val; V),        Leucine (Leu; L), Isoleucine (Ile; I), Proline (Pro; P),        Tryptophan (Trp; W), Phenylalanine (Phe; F), and Methionine        (Met, M).    -   2. Neutral Polar: Glycine (Gly; G); Serine (Ser; S), Threonine        (Thr; T), Tyrosine (Tyr; Y), Cysteine (Cys; C), Glutamine (Glu;        Q), Asparagine (Asn; N), and Norleucine.    -   3. Acidic: Aspartic Acid (Asp; D), Glutamic Acid (Glu; E);    -   4) Basic: Lysine (Lys; K), Arginine (Arg; R), Histidine (His;        H).        See Lewin, B., Genes V, Oxford University Press (1994), p. 11.

Conservative amino acid substitutions may encompass unconventional aminoacid residues, which are typically incorporated by chemical peptidesynthesis rather than by synthesis in biological systems. These include,without limitation, peptidomimetics and other reversed or inverted formsof amino acid moieties. Non-conservative substitutions may involve theexchange of a member of one of these classes for a member from anotherclass.

In making such changes, according to certain embodiments, thehydropathic index of amino acids may be considered. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics. They are: isoleucine (+4.5); valine (+4.2);leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is understood in the art.Kyte et al., J. Mol. Biol., 157:105-131 (1982). It is known that certainamino acids may be substituted for other amino acids having a similarhydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, in certainembodiments, the substitution of amino acids whose hydropathic indicesare within ±2 is included. In certain embodiments, those which arewithin ±1 are included, and in certain embodiments, those within ±0.5are included.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functional peptibody or peptidethereby created is intended for use in immunological embodiments, as inthe present case. In certain embodiments, the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigenicity, i.e., with a biological property of the protein.

The following hydrophilicity values have been assigned to these aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1);glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5);histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5)and tryptophan (−3.4). In making changes based upon similarhydrophilicity values, in certain embodiments, the substitution of aminoacids whose hydrophilicity values are within ±2 is included, in certainembodiments, those which are within ±1 are included, and in certainembodiments, those within ±0.5 are included. One may also identifyepitopes from primary amino acid sequences on the basis ofhydrophilicity. These regions are also referred to as “epitopic coreregions.”

Exemplary amino acid substitutions are set forth in Table 11 below.

TABLE 11 Amino Acid Substitutions Original Residues ExemplarySubstitutions Preferred Substitutions Ala Val, Leu, Ile Val Arg Lys,Gln, Asn Lys Asn Gln, Glu, Asp Gln Asp Glu, Gln, Asp Glu Cys Ser, AlaSer Gln Asn, Glu, Asp Asn Glu Asp, Gln, Asn Asp Gly Pro, Ala Ala HisAsn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Phe, Leu Norleucine LeuNorleucine, Ile, Val, Met, Ile Ala, Phe Lys Arg, 1,4 Diamino-butyric ArgAcid, Gln, Asn Met Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Tyr Leu ProAla Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe,Thr, Ser Phe Val Ile, Met, Leu, Phe, Ala, Leu Norleucine

A skilled artisan will be able to determine suitable variants of usefulpolypeptide or peptides as set forth herein using well-known techniques.In certain embodiments, one skilled in the art may identify suitableareas of the molecule that may be changed without destroying activity bytargeting regions not believed to be important for activity. In certainembodiments, one can identify residues and portions of the moleculesthat are conserved among similar peptides or polypeptides. In certainembodiments, one may even subject areas important for biologicalactivity or for structure to conservative amino acid substitutionswithout destroying the biological activity or without adverselyaffecting the polypeptide structure.

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar polypeptides that are importantfor activity or structure. In view of such a comparison, one can predictthe importance of amino acid residues in a protein that correspond toamino acid residues which are important for activity or structure insimilar proteins. One skilled in the art may opt for chemically similaramino acid substitutions for such predicted important amino acidresidues.

One skilled in the art can also analyze the three-dimensional structureand amino acid sequence in relation to that structure in similarpolypeptides. In view of such information, one skilled in the art maypredict the alignment of amino acid residues of an antibody with respectto its three dimensional structure. In certain embodiments, one skilledin the art may choose not to make radical changes to amino acid residuespredicted to be on the surface of the protein, since such residues maybe involved in important interactions with other molecules. Moreover,one skilled in the art may generate test variants containing a singleamino acid substitution at each desired amino acid residue. The variantscan then be screened using activity assays known to those skilled in theart. Such variants could be used to gather information about suitablevariants. For example, if one discovered that a change to a particularamino acid residue resulted in destroyed, undesirably reduced, orunsuitable activity, variants with such a change may be avoided. Inother words, based on information gathered from such routineexperiments, one skilled in the art can readily determine the aminoacids where further substitutions should be avoided either alone or incombination with other mutations.

A number of scientific publications have been devoted to the predictionof secondary structure. See Moult J., Curr. Op. in Biotech.,7(4):422-427 (1996), Chou et al., Biochemistry, 13(2):222-245 (1974);Chou et al., Biochemistry, 113(2):211-222 (1974); Chou et al., Adv.Enzymol. Relat. Areas Mol. Biol., 47:45-148 (1978); Chou et al., Ann.Rev. Biochem., 47:251-276 and Chou et al., Biophys. J., 26:367-384(1979). Moreover, computer programs are currently available to assistwith predicting secondary structure. One method of predicting secondarystructure is based upon homology modeling. For example, two polypeptidesor proteins that have a sequence identity of greater than 30%, orsimilarity greater than 40% often have similar structural topologies.The recent growth of the protein structural database (PDB) has providedenhanced predictability of secondary structure, including the potentialnumber of folds within a polypeptide's or protein's structure. See Holmet al., Nucl. Acid. Res., 27(1): 244-247 (1999). It has been suggested(Brenner et al., Curr. Op. Struct. Biol., 7(3): 369-376 (1997)) thatthere are a limited number of folds in a given polypeptide or proteinand that once a critical number of structures have been resolved,structural prediction will become dramatically more accurate.

Additional methods of predicting secondary structure include “threading”(Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87 (1997); Sippl et al.,Structure, 4(1):15-19 (1996)), “profile analysis” (Bowie et al.,Science, 253:164-170 (1991); Gribskov et al., Meth. Enzym., 183:146-159(1990); Gribskov et al., Proc. Nat. Acad. Sci., 84(13):4355-4358(1987)), and “evolutionary linkage” (See Holm, supra (1999), andBrenner, supra (1997)).

In certain embodiments, peptibody variants include glycosylationvariants wherein one or more glycosylation sites, such as a N-linkedglycosylation site, has been added to the peptibody. An N-linkedglycosylation site is characterized by the sequence: Asn-X-Ser orAsn-X-Thr, wherein the amino acid residue designated as X may be anyamino acid residue except proline. The substitution or addition of aminoacid residues to create this sequence provides a potential new site forthe addition of an N-linked carbohydrate chain. Alternatively,substitutions which eliminate this sequence will remove an existingN-linked carbohydrate chain. Also provided is a rearrangement ofN-linked carbohydrate chains wherein one or more N-linked glycosylationsites (typically those that are naturally occurring) are eliminated andone or more new N-linked sites are created.

Compounds of the present invention may be changed at the DNA level, aswell. The DNA sequence of any portion of the compound may be changed tocodons more compatible with the chosen host cell. For E. coli, which isthe preferred host cell, optimized codons are known in the art. Codonsmay be substituted to eliminate restriction sites or to include silentrestriction sites, which may aid in processing of the DNA in theselected host cell. The Fc domain-, linker-, polypeptide-, and/orpeptide-encoding DNA sequences may be modified to include any of theforegoing sequence changes. Thus, all modifications, substitution,derivitizations, etc. discussed herein apply equally to all polypeptideor peptide portions of the inventive composition of matter.

One embodiment of the present invention includes “affinity matured”peptides and polypeptide portions, including peptibody embodiments. Thisprocedure contemplates increasing the affinity or the bio-activity ofthe peptides and ppolypeptides using phage display or other selectiontechnologies. Based on a consensus sequence (which is generated for acollection of related peptides), directed secondary phage displaylibraries can be generated in which the “core” amino acids (determinedfrom the consensus sequence) are held constant or are biased infrequency of occurrence. Alternatively, an individual peptide sequencecan be used to generate a biased, directed phage display library.Panning of such libraries can yield peptides (which can be converted topeptibodies) with enhanced binding to the target or with enhancedbio-activity.

Non-Peptide Analogs/Protein Mimetics.

Furthermore, non-peptide analogs of peptides that provide a stabilizedstructure or lessened biodegradation, are also useful. Peptide mimeticanalogs can be prepared based on a selected inhibitory peptide byreplacement of one or more residues by nonpeptide moieties. Preferably,the nonpeptide moieties permit the peptide to retain its naturalconfirmation, or stabilize a preferred, e.g., bioactive, confirmationwhich retains the ability to recognize and bind Ang-2. In one aspect,the resulting analog/mimetic exhibits increased binding affinity forAng-2. One example of methods for preparation of nonpeptide mimeticanalogs from peptides is described in Nachman et al., Regul. Pept.57:359-370 (1995). If desired, the peptides of the invention can bemodified, for instance, by glycosylation, amidation, carboxylation, orphosphorylation, or by the creation of acid addition salts, amides,esters, in particular C-terminal esters, and N-acyl derivatives of thepeptides of the invention. The peptibodies also can be modified tocreate peptide derivatives by forming covalent or noncovalent complexeswith other moieties. Covalently-bound complexes can be prepared bylinking the chemical moieties to functional groups on the side chains ofamino acids comprising the peptibodies, or at the N- or C-terminus

The peptides can be conjugated to a reporter group, including, but notlimited to a radiolabel, a fluorescent label, an enzyme (e.g., thatcatalyzes a colorimetric or fluorometric reaction), a substrate, a solidmatrix, or a carrier (e.g., biotin or avidin). The invention accordinglyprovides a molecule comprising a peptibody molecule, wherein themolecule preferably further comprises a reporter group selected from thegroup consisting of a radiolabel, a fluorescent label, an enzyme, asubstrate, a solid matrix, and a carrier. Such labels are well known tothose of skill in the art, e.g., biotin labels are particularlycontemplated. The use of such labels is well known to those of skill inthe art and is described in, e.g., U.S. Pat. Nos. 3,817,837; 3,850,752;3,996,345; and 4,277,437. Other labels that will be useful include butare not limited to radioactive labels, fluorescent labels andchemiluminescent labels. U.S. Patents concerning use of such labelsinclude, for example, U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;and 3,996,345. Any of the peptibodies of the present invention maycomprise one, two, or more of any of these labels.

Recombinant Methods.

In general, the peptide or polypeptide portions of the inventivecompounds of this invention (including peptides or polypeptides asadditional moieties, linkers, and/or Fc domains) largely can be made intransformed host cells using recombinant DNA techniques. To do so, arecombinant DNA molecule coding for the peptide is prepared. Methods ofpreparing such DNA molecules are well known in the art. For instance,sequences coding for the peptides could be excised from DNA usingsuitable restriction enzymes. Alternatively, the DNA molecule could besynthesized using chemical synthesis techniques, such as thephosphoramidate method. Also, a combination of these techniques could beused.

Accordingly, the present invention also relates to nucleic acids,expression vectors and host cells useful in producing polypeptidecompositions of the present invention. Host cells can be eukaryoticcells, with mammalian cells preferred, e.g., CHO cells and HEK293 cells.Host cells can also be prokaryotic cells, with E. coli cells mostpreferred.

The compounds of this invention largely can be made in transformed hostcells using recombinant DNA techniques. To do so, a recombinant DNAmolecule coding for the peptide is prepared. Methods of preparing suchDNA molecules are well known in the art. For instance, sequences codingfor the peptides could be excised from DNA using suitable restrictionenzymes. Alternatively, the DNA molecule could be synthesized usingchemical synthesis techniques, such as the phosphoramidate method. Also,a combination of these techniques could be used.

The invention also includes a vector capable of expressing the peptidesin an appropriate host. The vector comprises the DNA molecule that codesfor the peptides operatively linked to appropriate expression controlsequences. Methods of effecting this operative linking, either before orafter the DNA molecule is inserted into the vector, are well known.Expression control sequences include promoters, activators, enhancers,operators, ribosomal binding sites, start signals, stop signals, capsignals, polyadenylation signals, and other signals involved with thecontrol of transcription or translation.

The resulting vector having the DNA molecule thereon is used totransform an appropriate host. This transformation can be performedusing methods well known in the art.

Any of a large number of available and well-known host cells can be usedin the practice of this invention. The selection of a particular host isdependent upon a number of factors recognized by the art. These include,for example, compatibility with the chosen expression vector, toxicityof the peptides encoded by the DNA molecule, rate of transformation,ease of recovery of the peptides, expression characteristics, bio-safetyand costs. A balance of these factors must be struck with theunderstanding that not all host cells can be equally effective for theexpression of a particular DNA sequence. Within these generalguidelines, useful microbial host cells include bacteria (such as E.coli sp.), yeast (such as Saccharomyces sp.) and other fungi, insects,plants, mammalian (including human) cells in culture, or other hostsknown in the art.

Next, the transformed host cell is cultured and purified. Host cells canbe cultured under conventional fermentation conditions so that thedesired compounds are expressed. Such fermentation conditions are wellknown in the art. Finally, the peptides are purified from culture bymethods well known in the art.

The compounds can also be made by synthetic methods. Solid phasesynthesis is the preferred technique of making individual peptides sinceit is the most cost-effective method of making small peptides. Forexample, well known solid phase synthesis techniques include the use ofprotecting groups, linkers, and solid phase supports, as well asspecific protection and deprotection reaction conditions, linkercleavage conditions, use of scavengers, and other aspects of solid phasepeptide synthesis. Suitable techniques are well known in the art. (E.g.,Merrifield (1973), Chem. Polypeptides, pp. 335-61 (Katsoyannis andPanayotis eds.); Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis etal. (1985), Biochem. Intl. 10: 394-414; Stewart and Young (1969), SolidPhase Peptide Synthesis; U.S. Pat. No. 3,941,763; Finn et al. (1976),The Proteins (3rd ed.) 2: 105-253; and Erickson et al. (1976), TheProteins (3rd ed.) 2: 257-527; “Protecting Groups in Organic Synthesis,”3rd Edition, T. W. Greene and P. G. M. Wuts, Eds., John Wiley & Sons,Inc., 1999; NovaBiochem Catalog, 2000; “Synthetic Peptides, A User'sGuide,” G. A. Grant, Ed., W.H. Freeman & Company, New York, N.Y., 1992;“Advanced Chemtech Handbook of Combinatorial & Solid Phase OrganicChemistry,” W. D. Bennet, J. W. Christensen, L. K. Hamaker, M. L.Peterson, M. R. Rhodes, and H. H. Saneii, Eds., Advanced Chemtech, 1998;“Principles of Peptide Synthesis, 2nd ed.,” M. Bodanszky, Ed.,Springer-Verlag, 1993; “The Practice of Peptide Synthesis, 2nd ed.,” M.Bodanszky and A. Bodanszky, Eds., Springer-Verlag, 1994; “ProtectingGroups,” P. J. Kocienski, Ed., Georg Thieme Verlag, Stuttgart, Germany,1994; “Fmoc Solid Phase Peptide Synthesis, A Practical Approach,” W. C.Chan and P. D. White, Eds., Oxford Press, 2000, G. B. Fields et al.,Synthetic Peptides: A User's Guide, 1990, 77-183).

Whether the compositions of the present invention are prepared bysynthetic or recombinant techniques, suitable protein purificationtechniques can also be involved, when applicable. In some embodiments ofthe compositions of the invention, the toxin peptide portion and/or thehalf-life extending portion, or any other portion, can be prepared toinclude a suitable isotopic label (e.g., ¹²⁵I, ¹⁴C, ¹³C, ³⁵S, ³H, ²H,¹³N, ¹⁵N, ¹⁸O, ¹⁷O etc.), for ease of quantification or detection.

Compounds that contain derivatized peptides or polypeptides, or whichcontain non-peptide groups can be synthesized by well-known organicchemistry techniques.

Uses of the Inventive Compounds

In General.

The compounds of this invention have pharmacologic activity resultingfrom their ability to bind to proteins of interest as agonists, mimeticsor antagonists of the native ligands of such proteins of interest. Byway of example, the utility of a variety of specific compounds is shownin Tables 5-10. The activity of these compounds can be measured byassays known in the art.

In addition to therapeutic uses, the compounds of the present inventionare useful in diagnosing diseases characterized by dysfunction of theirassociated protein of interest. For some of these diagnostic embodimentsand for other detection (including semi-quantitative and quantitative)purposes, conjugation of the Fc domain to an immobilized substrate as anadditional functional moiety, such as but not limited to, a platesurface, a chip, a bead, a matrix or a particle, can be useful. Also amoiety detectably labeled with a radioisotope, an enzyme (e.g., aperoxidase or a kinase), a biotinyl moiety, a fluorophore, or achromophore can be useful for such purposes.

In one embodiment, a method of detecting in a biological sample aprotein of interest (e.g., a receptor) that is capable of beingactivated comprising the steps of: (a) contacting the sample with acompound of this invention; and (b) detecting activation of the proteinof interest by the compound. The biological samples include tissuespecimens, intact cells, or extracts thereof. The compounds of thisinvention may be used as part of a kit to detect the presence of theirassociated proteins of interest in a biological sample. Such kits employthe compounds of the invention having an attached label to allow fordetection. The compounds are useful for identifying normal or abnormalproteins of interest. For the EPO-mimetic compounds, for example,presence of abnormal protein of interest in a biological sample may beindicative of such disorders as Diamond Blackfan anemia, where it isbelieved that the EPO receptor is dysfunctional.

Therapeutic Uses of EPO-Mimetic Molecules

The EPO-mimetic compounds of the invention are useful for treatingdisorders characterized by low red blood cell levels. Included in theinvention are methods of modulating the endogenous activity of an EPOreceptor in a mammal, preferably methods of increasing the activity ofan EPO receptor. In general, any condition treatable by erythropoietin,such as anemia, may also be treated by the EPO-mimetic compounds of theinvention. These compounds are administered by an amount and route ofdelivery that is appropriate for the nature and severity of thecondition being treated and may be ascertained by one skilled in theart. Preferably, administration is by injection, either subcutaneous,intramuscular, or intravenous.

Therapeutic Uses of TPO-Mimetic Compounds

For the TPO-mimetic compounds, one can utilize such standard assays asthose described in WO95/26746 entitled “Compositions and Methods forStimulating Megakaryocyte Growth and Differentiation.” The conditions tobe treated are generally those that involve an existingmegakaryocyte/platelet deficiency or an expected megakaryocyte/plateletdeficiency (e.g., because of planned surgery or platelet donation). Suchconditions will usually be the result of a deficiency (temporary orpermanent) of active Mpl ligand in vivo. The generic term for plateletdeficiency is thrombocytopenia, and hence the methods and compositionsof the present invention are generally available for treatingthrombocytopenia in patients in need thereof.

Thrombocytopenia (platelet deficiencies) may be present for variousreasons, including chemotherapy and other therapy with a variety ofdrugs, radiation therapy, surgery, accidental blood loss, and otherspecific disease conditions. Exemplary specific disease conditions thatinvolve thrombocytopenia and may be treated in accordance with thisinvention are: aplastic anemia, idiopathic thrombocytopenia, metastatictumors which result in thrombocytopenia, systemic lupus erythematosus,splenomegaly, Fanconi's syndrome, vitamin B12 deficiency, folic aciddeficiency, May-Hegglin anomaly, Wiskott-Aldrich syndrome, andparoxysmal nocturnal hemoglobinuria. Also, certain treatments for AIDSresult in thrombocytopenia (e.g., AZT). Certain wound healing disordersmight also benefit from an increase in platelet numbers.

With regard to anticipated platelet deficiencies, e.g., due to futuresurgery, a compound of the present invention could be administeredseveral days to several hours prior to the need for platelets. Withregard to acute situations, e.g., accidental and massive blood loss, acompound of this invention could be administered along with blood orpurified platelets.

The TPO-mimetic compounds of this invention may also be useful instimulating certain cell types other than megakaryocytes if such cellsare found to express Mpl receptor. Conditions associated with such cellsthat express the Mpl receptor, which are responsive to stimulation bythe Mpl ligand, are also within the scope of this invention.

The TPO-mimetic compounds of this invention may be used in any situationin which production of platelets or platelet precursor cells is desired,or in which stimulation of the c-Mpl receptor is desired. Thus, forexample, the compounds of this invention may be used to treat anycondition in a mammal wherein there is a need of platelets,megakaryocytes, and the like. Such conditions are described in detail inthe following exemplary sources: WO95/26746; WO95/21919; WO95/18858;WO95/21920 and are incorporated herein.

The TPO-mimetic compounds of this invention may also be useful inmaintaining the viability or storage life of platelets and/ormegakaryocytes and related cells. Accordingly, it could be useful toinclude an effective amount of one or more such compounds in acomposition containing such cells.

Therapeutic Uses of Ang-2 Binding Molecules

Agents that modulate Ang-2 binding activity, or other cellular activity,may be used in combination with other therapeutic agents to enhancetheir therapeutic effects or decrease potential side effects.

In one aspect, the present invention provides reagents and methodsuseful for treating diseases and conditions characterized by undesirableor aberrant levels of Ang-2 activity in a cell. These diseases includecancers, and other hyperproliferative conditions, such as hyperplasia,psoriasis, contact dermatitis, immunological disorders, and infertility.

The present invention also provides methods of treating cancer in ananimal, including humans, comprising administering to the animal aneffective amount of a specific binding agent, such as a peptibody, thatinhibits or decreases Ang-2 activity. The invention is further directedto methods of inhibiting cancer cell growth, including processes ofcellular proliferation, invasiveness, and metastasis in biologicalsystems. Methods include use of a compound of the invention as aninhibitor of cancer cell growth. Preferably, the methods are employed toinhibit or reduce cancer cell growth, invasiveness, metastasis, or tumorincidence in living animals, such as mammals. Methods of the inventionare also readily adaptable for use in assay systems, e.g., assayingcancer cell growth and properties thereof, as well as identifyingcompounds that affect cancer cell growth.

The cancers treatable by methods of the present invention preferablyoccur in mammals. Mammals include, for example, humans and otherprimates, as well as pet or companion animals such as dogs and cats,laboratory animals such as rats, mice and rabbits, and farm animals suchas horses, pigs, sheep, and cattle.

Tumors or neoplasms include growths of tissue cells in which themultiplication of the cells is uncontrolled and progressive. Some suchgrowths are benign, but others are termed malignant and may lead todeath of the organism. Malignant neoplasms or cancers are distinguishedfrom benign growths in that, in addition to exhibiting aggressivecellular proliferation, they may invade surrounding tissues andmetastasize. Moreover, malignant neoplasms are characterized in thatthey show a greater loss of differentiation (greater dedifferentiation),and of their organization relative to one another and their surroundingtissues. This property is also called “anaplasia.”

Neoplasms treatable by the present invention also include solid tumors,i.e., carcinomas and sarcomas. Carcinomas include those malignantneoplasms derived from epithelial cells that infiltrate (invade) thesurrounding tissues and give rise to metastases. Adenocarcinomas arecarcinomas derived from glandular tissue, or which form recognizableglandular structures. Another broad category or cancers includessarcomas, which are tumors whose cells are embedded in a fibrillar orhomogeneous substance like embryonic connective tissue. The inventionalso enables treatment of cancers of the myeloid or lymphoid systems,including leukemias, lymphomas and other cancers that typically do notpresent as a tumor mass, but are distributed in the vascular orlymphoreticular systems.

The ang-2 binding molecules of this invention are thus useful for thetreatment of a wide variety of cancers, including solid tumors andleukemias. Types of cancer or tumor cells amenable to treatmentaccording to the invention include, for example, ACTH-producing tumor;acute lymphocytic leukemia; acute nonlymphocytic leukemia; adenoma;cancer of the adrenal cortex; adenocarcinoma of the breast, prostate,and colon; ameloblastoma; apudoma; bladder cancer; brain cancer;branchioma; breast cancer; all forms of bronchogenic carcinoma of thelung; carcinoid heart disease; carcinoma (e.g., Walker, basal cell,basosquamous, Brown-Pearce, ductal, Ehrlich tumor, Krebs 2, merkel cell,mucinous, non-small cell lung, oat cell, papillary, scirrhous,bronchiolar, bronchogenic, squamous cell, and transitional cell);malignant carcinoid syndrome; immunoproliferative small lung cellcarcinoma; cementoma; cervical cancer; chondroblastoma; chondroma;chondrosarcoma; choristoma; chronic lymphocytic leukemia; chronicmyelocytic leukemia; colorectal cancer; chordoma; craniopharyngioma;cutaneous T-cell lymphoma; dysgerminoma; endometrial cancer; esophagealcancer; Ewing's sarcoma; fibroma; fibrosarcoma; gallbladder cancer;giant cell tumors; glioma; hairy cell leukemia; hamartoma; head and neckcancer; hepatoma; histiocytic disorders; histiocytosis; Hodgkin'slymphoma; Kaposi's sarcoma; kidney cancer; lipoma; liposarcoma; livercancer; lung cancer (small and non-small cell); malignant peritonealeffusion; malignant pleural effusion; melanoma; mesenchymoma;mesonephroma; mesothelioma; multiple myeloma; myosarcoma; myxoma;myxosarcoma; neuroblastoma; non-Hodgkin's lymphoma; odontoma; osteoma;osteosarcoma; ovarian cancer; ovarian (germ cell) cancer; pancreaticcancer; papilloma; penile cancer; plasmacytoma; prostate cancer;reticuloendotheliosis; retinoblastoma; skin cancer; soft tissue sarcoma;squamous cell carcinomas; stomach cancer; teratoma; testicular cancer;thymoma; thyroid cancer; trophoblastic neoplasms; uterine cancer;vaginal cancer; cancer of the vulva; Wilms' tumor.

Further, the following types of cancers may also be treated:cholangioma; cholesteatoma; cyclindroma; cystadenocarcinoma;cystadenoma; granulosa cell tumor; gynandroblastoma; hidradenoma; isletcell tumor; Leydig cell tumor; papilloma; Sertoli cell tumor; theca celltumor; leiomyoma; leiomyosarcoma; myoblastoma; myoma; myosarcoma;rhabdomyoma; rhabdomyosarcoma; ependymoma; ganglioneuroma; glioma;medulloblastoma; meningioma; neurilemmoma; neuroblastoma;neuroepithelioma; neurofibroma; neuroma; paraganglioma; paragangliomanonchromaffin; angiokeratoma; angiolymphoid hyperplasia witheosinophilia; angioma sclerosing; angiomatosis; glomangioma;hemangioendothelioma; hemangioma; hemangiopericytoma; hemangiosarcoma;lymphangioma; lymphangiomyoma; lymphangiosarcoma; pinealoma;carcinosarcoma; chondrosarcoma; cystosarcoma phyllodes; fibrosarcoma;hemangiosarcoma; leiomyosarcoma; leukosarcoma; liposarcoma;lymphangiosarcoma; myosarcoma; myxosarcoma; ovarian carcinoma;rhabdomyosarcoma; sarcoma; neoplasms; nerofibromatosis; and cervicaldysplasia.

Therapeutic Uses of NGF Binding Molecules

The NGF binding molecules may be used in the prevention or treatment ofNGF-related diseases and disorders. Such indications include but are notlimited to pain (including, but not limited to, inflammatory pain andassociated hyperalgesia and allodynia, neuropathic pain and associatedhyperalgesia and allodynia, diabetic neuropathy pain, causalgia,sympathetically maintained pain, deafferentation syndromes, acute pain,tension headache, migraine, dental pain, pain from trauma, surgicalpain, pain resulting from amputation or abscess, causalgia,demyelinating diseases, and trigeminal neuralgia). The peptides andmodified peptides of the invention have therapeutic value for theprevention or treatment of other diseases linked to NGF as a causativeagent, including, but not limited to, asthma, urge incontinence (i.e.,hyperactive bladder), psoriasis, cancer (especially, pancreatic cancerand melanoma), chronic alcoholism, stroke, thalamic pain syndrome,diabetes, acquired immune deficiency syndrome (“AIDS”), toxins andchemotherapy, general headache, migraine, cluster headache,mixed-vascular and non-vascular syndromes, general inflammation,arthritis, rheumatic diseases, lupus, osteoarthritis, inflammatory boweldisorders, inflammatory eye disorders, inflammatory or unstable bladderdisorders, psoriasis, skin complaints with inflammatory components,sunburn, carditis, dermatitis, myositis, neuritis, collagen vasculardiseases, chronic inflammatory conditions, asthma, epithelial tissuedamage or dysfunction, herpes simplex, disturbances of visceral motilityat respiratory, genitourinary, gastrointestinal or vascular regions,wounds, burns, allergic skin reactions, pruritis, vitiligo, generalgastrointestinal disorders, colitis, gastric ulceration, duodenalulcers, vasomotor or allergic rhinitis, or bronchial disorders.

Therapeutic Uses of Myostatin Binding Molecules

The myostatin binding agents of the present invention bind to myostatinand block or inhibit myostatin signaling within targeted cells. Thepresent invention provides methods and reagents for reducing the amountor activity of myostatin in an animal by administering an effectivedosage of one or more myostatin binding agents to the animal. In oneaspect, the present invention provides methods and reagents for treatingmyostatin-related disorders in an animal comprising administering aneffective dosage of one or more binding agents to the animal. Thesemyostatin-related disorders include but are not limited to various formsof muscle wasting, as well as metabolic disorders such as diabetes andrelated disorders, and bone degenerative diseases such as osteoporosis.

As shown in the Example 8 of U.S. Ser. No. 10/742,379, exemplarypeptibodies of the present invention dramatically increases lean musclemass in the CD1 nu/nu mouse model. This in vivo activity correlates tothe in vitro binding and inhibitory activity described below for thesame peptibodies.

Muscle wasting disorders include dystrophies such as Duchenne's musculardystrophy, progressive muscular dystrophy, Becker's type musculardystrophy, Dejerine-Landouzy muscular dystrophy, Erb's musculardystrophy, and infantile neuroaxonal muscular dystrophy. For example,blocking myostatin through use of antibodies in vivo improved thedystrophic phenotype of the mdx mouse model of Duchenne musculardystrophy (Bogdanovich et al. (2002), Nature 420: 28). Use of anexemplary peptibody increases lean muscle mass and increases the ratioof lean muscle to fat in mdx mouse models as described in Example 9below.

Additional muscle wasting disorders arise from chronic disease such asamyotrophic lateral sclerosis, congestive obstructive pulmonary disease,cancer, AIDS, renal failure, and rheumatoid arthritis. For example,cachexia or muscle wasting and loss of body weight was induced inathymic nude mice by a systemically administered myostatin (Zimmers etal., supra). In another example, serum and intramuscular concentrationsof myostatin-immunoreactive protein was found to be increased in menexhibiting AIDS-related muscle wasting and was inversely related tofat-free mass (Gonzalez-Cadavid et al. (1998), PNAS USA 95:14938-14943). Additional conditions resulting in muscle wasting mayarise from inactivity due to disability such as confinement in awheelchair, prolonged bedrest due to stroke, illness, bone fracture ortrauma, and muscular atrophy in a microgravity environment (spaceflight). For example, plasma myostatin immunoreactive protein was foundto increase after prolonged bedrest (Zachwieja et al. J Gravit Physiol.6(2):11(1999). It was also found that the muscles of rats exposed to amicrogravity environment during a space shuttle flight expressed anincreased amount of myostatin compared with the muscles of rats whichwere not exposed (Lalani et al. (2000), J. Endocrin. 167(3):417-28).

In addition, age-related increases in fat to muscle ratios, andage-related muscular atrophy appear to be related to myostatin. Forexample, the average serum myostatin-immunoreactive protein increasedwith age in groups of young (19-35 yr old), middle-aged (36-75 yr old),and elderly (76-92 yr old) men and women, while the average muscle massand fat-free mass declined with age in these groups (Yarasheski et al. JNutr Aging 6(5):343-8 (2002)). It has also been shown that myostatingene knockout in mice increased myogenesis and decreased adipogenesis(Lin et al. (2002), Biochem Biophys Res Commun 291(3):701-6, resultingin adults with increased muscle mass and decreased fat accumulation andleptin secretion. Exemplary molecules improve the lean muscle mass tofat ratio in aged mdx mice as shown below.

In addition, myostatin has now been found to be expressed at low levelsin heart muscle and expression is upregulated after cardiomyocytes afterinfarct (Sharma et al. (1999), J Cell Physiol. 180(1):1-9). Therefore,reducing myostatin levels in the heart muscle may improve recovery ofheart muscle after infarct.

Myostatin also appears to influence metabolic disorders including type 2diabetes, noninsulin-dependent diabetes mellitus, hyperglycemia, andobesity. For example, lack of myostatin has been shown to improve theobese and diabetic phenotypes of two mouse models (Yen et al. supra). Inaddition, increasing muscle mass by reducing myostatin levels mayimprove bone strength and reduce osteoporosis and other degenerativebone diseases. It has been found, for example, that myostatin-deficientmice showed increased mineral content and density of the mouse humerusand increased mineral content of both trabecular and cortical bone atthe regions where the muscles attach, as well as increased muscle mass(Hamrick et al. (2002), Calcif Tissue Int 71(1): 63-8). In the presentinvention, an exemplary peptibody increases the lean muscle mass to fatratio in mdx mouse models as shown below.

The present invention also provides methods and reagents for increasingmuscle mass in food animals by administering an effective dosage of themyostatin binding agent to the animal. Since the mature C-terminalmyostatin polypeptide is identical in all species tested, myostatinbinding agents would be expected to be effective for increasing musclemass and reducing fat in any agriculturally important species includingcattle, chicken, turkeys, and pigs.

The myostatin-binding molecules of the present invention may be usedalone or in combination with other therapeutic agents to enhance theirtherapeutic effects or decrease potential side effects. The molecules ofthe present invention possess one or more desirable but unexpectedcombination of properties to improve the therapeutic value of theagents. These properties include increased activity, increasedsolubility, reduced degradation, increased half-life, reduced toxicity,and reduced immunogenicity. Thus the molecules of the present inventionare useful for extended treatment regimes. In addition, the propertiesof hydrophilicity and hydrophobicity of the compounds of the inventionare well balanced, thereby enhancing their utility for both in vitro andespecially in vivo uses. Specifically, compounds of the invention havean appropriate degree of solubility in aqueous media that permitsabsorption and bioavailability in the body, while also having a degreeof solubility in lipids that permits the compounds to traverse the cellmembrane to a putative site of action, such as a particular muscle mass.

The myostatin-binding molecules of the present invention are useful fortreating a “subject” or any animal, including humans, when administeredin an effective dosages in a suitable composition.

In addition, the mystatin-binding molecules of the present invention areuseful for detecting and quantitating myostatin in a number of assays.These assays are described in detail in U.S. Ser. No. 10/742,379.

In general, the myostatin-binding molecules of the present invention areuseful as capture agents to bind and immobilize myostatin in a varietyof assays, similar to those described, for example, in Asai, ed.,Methods in Cell Biology, 37, Antibodies in Cell Biology, Academic Press,Inc., New York (1993). The myostatin-binding molecule may be labeled insome manner or may react with a third molecule such as an anti-bindingmolecule antibody which is labeled to enable myostatin to be detectedand quantitated. For example, a myostatin-binding molecule or a thirdmolecule can be modified with a detectable moiety, such as biotin, whichcan then be bound by a fourth molecule, such as enzyme-labeledstreptavidin, or other proteins. (Akerstrom (1985), J Immunol 135:2589;Chaubert (1997), Mod Pathol 10:585).

Throughout any particular assay, incubation and/or washing steps may berequired after each combination of reagents. Incubation steps can varyfrom about 5 seconds to several hours, preferably from about 5 minutesto about 24 hours. However, the incubation time will depend upon theassay format, volume of solution, concentrations, and the like. Usually,the assays will be carried out at ambient temperature, although they canbe conducted over a range of temperatures.

Therapeutic Uses of BAFF-Binding Molecules.

BAFF-binding molecules of this invention may be particularly useful intreatment of B-cell mediated autoimmune diseases. In particular, theymay be useful in treating, preventing, ameliorating, diagnosing orprognosing lupus, including systemic lupus erythematosus (SLE), andlupus-associated diseases and conditions. Other preferred indicationsinclude B-cell mediated cancers, including B-cell lymphoma.

The compounds of this invention can also be used to treat inflammatoryconditions of the joints. Inflammatory conditions of a joint are chronicjoint diseases that afflict and disable, to varying degrees, millions ofpeople worldwide. Rheumatoid arthritis is a disease of articular jointsin which the cartilage and bone are slowly eroded away by aproliferative, invasive connective tissue called pannus, which isderived from the synovial membrane. The disease may involveperi-articular structures such as bursae, tendon sheaths and tendons aswell as extra-articular tissues such as the subcutis, cardiovascularsystem, lungs, spleen, lymph nodes, skeletal muscles, nervous system(central and peripheral) and eyes (Silberberg (1985), Anderson'sPathology, Kissane (ed.), II:1828). Osteoarthritis is a common jointdisease characterized by degenerative changes in articular cartilage andreactive proliferation of bone and cartilage around the joint.Osteoarthritis is a cell-mediated active process that may result fromthe inappropriate response of chondrocytes to catabolic and anabolicstimuli. Changes in some matrix molecules of articular cartilagereportedly occur in early osteoarthritis (Thonar et al. (1993),Rheumatic disease clinics of North America, Moskowitz (ed.), 19:635-657and Shinmei et al. (1992), Arthritis Rheum., 35:1304-1308). TALL-1,TALL-1R and modulators thereof are believed to be useful in thetreatment of these and related conditions.

BAFF-binding molecules may also be useful in treatment of a number ofadditional diseases and disorders, including acute pancreatitis; ALS;Alzheimer's disease; asthma; atherosclerosis; autoimmune hemolyticanemia; cancer, particularly cancers related to B cells;cachexia/anorexia; chronic fatigue syndrome; cirrhosis (e.g., primarybiliary cirrhosis); diabetes (e.g., insulin diabetes); fever;glomerulonephritis, including IgA glomerulonephritis and primaryglomerulonephritis; Goodpasture's syndrome; Guillain-Barre syndrome;graft versus host disease; Hashimoto's thyroiditis; hemorrhagic shock;hyperalgesia; inflammatory bowel disease; inflammatory conditions of ajoint, including osteoarthritis, psoriatic arthritis and rheumatoidarthritis; inflammatory conditions resulting from strain, sprain,cartilage damage, trauma, orthopedic surgery, infection or other diseaseprocesses; insulin-dependent diabetes mellitus; ischemic injury,including cerebral ischemia (e.g., brain injury as a result of trauma,epilepsy, hemorrhage or stroke, each of which may lead toneurodegeneration); learning impairment; lung diseases (e.g., ARDS);lupus, particularly systemic lupus erythematosus (SLE); multiplemyeloma; multiple sclerosis; Myasthenia gravis; myelogenous (e.g., AMLand CML) and other leukemias; myopathies (e.g., muscle proteinmetabolism, esp. in sepsis); neurotoxicity (e.g., as induced by HIV);osteoporosis; pain; Parkinson's disease; Pemphigus;polymyositis/dermatomyositis; pulmonary inflammation, includingautoimmune pulmonary inflammation; pre-term labor; psoriasis; Reiter'sdisease; reperfusion injury; septic shock; side effects from radiationtherapy; Sjogren's syndrome; sleep disturbance; temporal mandibularjoint disease; thrombocytopenia, including idiopathic thrombocytopeniaand autoimmune neonatal thrombocytopenia; tumor metastasis; uveitis; andvasculitis.

Combination Therapy.

The therapeutic methods, compositions and compounds of the presentinvention may also be employed, alone or in combination with othercytokines, soluble Mpl receptor, hematopoietic factors, interleukins,growth factors or antibodies in the treatment of disease statescharacterized by other symptoms as well as platelet deficiencies. It isanticipated that the inventive compound will prove useful in treatingsome forms of thrombocytopenia in combination with general stimulatorsof hematopoiesis, such as IL-3 or GM-CSF. Other megakaryocyticstimulatory factors, i.e., meg-CSF, stem cell factor (SCF), leukemiainhibitory factor (LIF), oncostatin M (OSM), or other molecules withmegakaryocyte stimulating activity may also be employed with Mpl ligand.Additional exemplary cytokines or hematopoietic factors for suchco-administration include IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5,IL-6, IL-11, colony stimulating factor-1 (CSF-1), SCF, GM-CSF,granulocyte colony stimulating factor (G-CSF), EPO, interferon-alpha(IFN-alpha), consensus interferon, IFN-beta, or IFN-gamma. It mayfurther be useful to administer, either simultaneously or sequentially,an effective amount of a soluble mammalian Mpl receptor, which appearsto have an effect of causing megakaryocytes to fragment into plateletsonce the megakaryocytes have reached mature form. Thus, administrationof an inventive compound (to enhance the number of maturemegakaryocytes) followed by administration of the soluble Mpl receptor(to inactivate the ligand and allow the mature megakaryocytes to produceplatelets) is expected to be a particularly effective means ofstimulating platelet production. The dosage recited above would beadjusted to compensate for such additional components in the therapeuticcomposition. Progress of the treated patient can be monitored byconventional methods.

In cases where the inventive compounds are added to compositions ofplatelets and/or megakaryocytes and related cells, the amount to beincluded will generally be ascertained experimentally by techniques andassays known in the art. An exemplary range of amounts is 0.1 μg-1 mginventive compound per 10⁶ cells.

Therapeutics Incorporating Toxin Peptides.

Some embodiments of the inventive composition of matter incorporatetoxin peptides as additional functional moieties, which toxin peptidescan have pharmacologic activity resulting from the ability to bind toion channels of interest as agonists, mimetics or antagonists of thenative ligands of such ion channels of interest. Consequently suchembodiments of the inventive composition of matter can have utility inthe treatment of pathologies associated with ion channels. Heritablediseases that have a known linkage to ion channels (“channelopathies”)cover various fields of medicine, some of which include neurology,nephrology, myology and cardiology. A list of inherited disordersattributed to ion channels (channel types in parentheses) includes:

-   -   cystic fibrosis (Cl⁻ channel; CFTR),    -   Dent's disease (proteinuria and hypercalciuria; Cl⁻ channel;        CLCN5),    -   osteopetrosis (Cl⁻ channel; CLCN7),    -   familial hyperinsulinemia (SUR1; KCNJ11; K channel),    -   diabetes (KATP/SUR channel),    -   Andersen syndrome (KCNJ2, Kir2.1 K channel),    -   Bartter syndrome (KCNJ1; Kir1.1/ROMK; K channel),    -   hereditary hearing loss (KCNQ4; K channel),    -   hereditary hypertension (Liddle's syndrome; SCNN1; epithelial Na        channel),    -   dilated cardiomyopathy (SUR2, K channel),    -   long-QT syndrome or cardiac arrhythmias (cardiac potassium and        sodium channels),    -   Thymothy syndrome (CACNA1C, Cav1.2),    -   myasthenic syndromes (CHRNA, CHRNB, CNRNE; nAChR), and a variety        of other myopathies,    -   hyperkalemic periodic paralysis (Na and K channels),    -   epilepsy (Na⁺ and K⁺ channels),    -   hemiplegic migraine (CACNA1A, Cav2.1 Ca²⁺ channel and ATP1A2),    -   central core disease (RYR1, RyR1; Ca²⁺ channel), and    -   paramyotonia and myotonia (Na⁺, Cl⁻ channels)        See L. J. Ptacek and Y-H Fu (2004), Arch. Neurol. 61:        166-8; B. A. Niemeyer et al. (2001), EMBO reports 21: 568-73; F.        Lehmann-Horn and K. Jurkat-Rott (1999), Physiol. Rev. 79:        1317-72. Although the foregoing list concerned disorders of        inherited origin, molecules targeting the channels cited in        these disorders can also be useful in treating related disorders        of other, or indeterminate, origin.

In addition to the aforementioned disorders, evidence has also beenprovided supporting ion channels as targets for treatment of:

-   -   sickle cell anemia (IKCa1)—in sickle cell anemia, water loss        from erythrocytes leads to hemoglobin polymerization and        subsequent hemolysis and vascular obstruction. The water loss is        consequent to potassium efflux through the so-called Gardos        channel i.e., IKCa1. Therefore, block of IKCa1 is a potential        therapeutic treatment for sickle cell anemia.    -   glaucoma (BKCa), —in glaucoma the intraocular pressure is too        high leading to optic nerve damage, abnormal eye function and        possibly blindness. Block of BKCa potassium channels can reduce        intraocular fluid secretion and increase smooth muscle        contraction, possibly leading to lower intraocular pressure and        neuroprotection in the eye. multiple sclerosis (Kv, KCa),    -   psoriasis (Kv, KCa),    -   arthritis (Kv, KCa),    -   asthma (KCa, Kv),    -   allergy(KCa, Kv),    -   COPD (KCa, Kv, Ca),    -   allergic rhinitis (KCa, Kv),    -   pulmonary fibrosis,    -   lupus (IKCa1, Kv),    -   transplantation, GvHD (KCa, Kv),    -   inflammatory bone resorption (KCa, Kv),    -   periodontal disease (KCa, Kv),    -   diabetes, type I (Kv), —type I diabetes is an autoimmune disease        that is characterized by abnormal glucose, protein and lipid        metabolism and is associated with insulin deficiency or        resistance. In this disease, Kv1.3-expressing T-lymphocytes        attack and destroy pancreatic islets leading to loss of        beta-cells. Block of Kv1.3 decreases inflammatory cytokines. In        addition block of Kv1.3 facilitates the translocation of GLUT4        to the plasma membrane, thereby increasing insulin sensitivity.    -   obesity (Kv),—Kv1.3 appears to play a critical role in        controlling energy homeostasis and in protecting against        diet-induced obesity. Consequently, Kv1.3 blockers could        increase metabolic rate, leading to greater energy utilization        and decreased body weight.    -   restenosis (KCa, Ca²⁺), —proliferation and migration of vascular        smooth muscle cells can lead to neointimal thickening and        vascular restenosis. Excessive neointimal vascular smooth muscle        cell proliferation is associated with elevated expression of        IKCa1. Therefore, block of IKCa1 could represent a therapeutic        strategy to prevent restenosis after angioplasty.    -   ischaemia (KCa, Ca²⁺), —in neuronal or cardiac ischemia,        depolarization of cell membranes leads to opening of        voltage-gated sodium and calcium channels. In turn this can lead        to calcium overload, which is cytotoxic. Block of voltage-gated        sodium and/or calcium channels can reduce calcium overload and        provide cytoprotective effects. In addition, due to their        critical role in controlling and stabilizing cell membrane        potential, modulators of voltage- and calcium-activated        potassium channels can also act to reduce calcium overload and        protect cells.    -   renal incontinence (KCa), renal incontinence is associated with        overactive bladder smooth muscle cells. Calcium-activated        potassium channels are expressed in bladder smooth muscle cells,        where they control the membrane potential and indirectly control        the force and frequency of cell contraction. Openers of        calcium-activated potassium channels therefore provide a        mechanism to dampen electrical and contractile activity in        bladder, leading to reduced urge to urinate.    -   osteoporosis (Kv),    -   pain, including migraine (Na_(v), TRP [transient receptor        potential channels], P2X, Ca²⁺), N-type voltage-gated calcium        channels are key regulators of nociceptive neurotransmission in        the spinal cord. Ziconotide, a peptide blocker of N-type calcium        channels reduces nociceptive neurotransmission and is approved        worldwide for the symptomatic alleviation of severe chronic pain        in humans. Novel blockers of nociceptor-specific N-type calcium        channels would be improved analgesics with reduced side-effect        profiles.    -   hypertension (Ca²⁺), -L-type and T-type voltage-gated calcium        channels are expressed in vascular smooth muscle cells where        they control excitation-contraction coupling and cellular        proliferation. In particular, T-type calcium channel activity        has been linked to neointima formation during hypertension.        Blockers of L-type and T-type calcium channels are useful for        the clinical treatment of hypertension because they reduce        calcium influx and inhibit smooth muscle cell contraction.    -   wound healing, cell migration serves a key role in wound        healing. Intracellular calcium gradients have been implicated as        important regulators of cellular migration machinery in        keratinocytes and fibroblasts. In addition, ion flux across cell        membranes is associated with cell volume changes. By controlling        cell volume, ion channels contribute to the intracellular        environment that is required for operation of the cellular        migration machinery. In particular, IKCa1 appears to be required        universally for cell migration. In addition, Kv1.3, Kv3.1, NMDA        receptors and N-type calcium channels are associated with the        migration of lymphocytes and neurons.    -   stroke,    -   Alzheimer's,    -   Parkenson's Disease (nACHR, Nav)    -   Bipolar Disorder (Nav, Cav)    -   cancer, many potassium channel genes are amplified and protein        subunits are upregulated in many cancerous condition. Consistent        with a pathophysiological role for potassium channel        upregulation, potassium channel blockers have been shown to        suppress proliferation of uterine cancer cells and        hepatocarcinoma cells, presumably through inhibition of calcium        influx and effects on calcium-dependent gene expression.    -   a variety of neurological, cardiovascular, metabolic and        autoimmune diseases.

Both agonists and antagonists of ion channels can achieve therapeuticbenefit. Therapeutic benefits can result, for example, from antagonizingKv1.3, IKCa1, SKCa, BKCa, N-type or T-type Ca²⁺ channels and the like.Small molecule and peptide antagonists of these channels have been shownto possess utility in vitro and in vivo.

The diseases and pharmacologically active additional moieties describedherein are merely exemplary and in no way limit the range of inventivepharmacologically active compounds and compositions that can be preparedusing the inventive method or the diseases and disorders that can betreated with the benefit of the present invention.

Pharmaceutical Compositions

In General.

The present invention also provides pharmaceutical compositionscomprising the inventive composition of matter and a pharmaceuticallyacceptable carrier. Such pharmaceutical compositions can be configuredfor administration to a patient by a wide variety of delivery routes,e.g., an intravascular delivery route such as by injection or infusion,subcutaneous, intramuscular, intraperitoneal, epidural, or intrathecaldelivery routes, or for oral, enteral, pulmonary (e.g., inhalant),intranasal, transmucosal (e.g., sublingual administration), transdermalor other delivery routes and/or forms of administration known in theart. The inventive pharmaceutical compositions may be prepared in liquidform, or may be in dried powder form, such as lyophilized form. For oralor enteral use, the pharmaceutical compositions can be configured, forexample, as tablets, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules, emulsions, hard or soft capsules,syrups, elixirs or enteral formulas.

In the practice of this invention the “pharmaceutically acceptablecarrier” is any physiologically tolerated substance known to those ofordinary skill in the art useful in formulating pharmaceuticalcompositions, including, any pharmaceutically acceptable diluents,excipients, dispersants, binders, fillers, glidants, anti-frictionalagents, compression aids, tablet-disintegrating agents (disintegrants),suspending agents, lubricants, flavorants, odorants, sweeteners,permeation or penetration enhancers, preservatives, surfactants,solubilizers, emulsifiers, thickeners, adjuvants, dyes, coatings,encapsulating material(s), and/or other additives singly or incombination. Such pharmaceutical compositions can include diluents ofvarious buffer content (e.g., Tris-HCl, acetate, phosphate), pH andionic strength; additives such as detergents and solubilizing agents(e.g., Tween® 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid,sodium metabisulfite), preservatives (e.g., Thimersol®, benzyl alcohol)and bulking substances (e.g., lactose, mannitol); incorporation of thematerial into particulate preparations of polymeric compounds such aspolylactic acid, polyglycolic acid, etc. or into liposomes. Hyaluronicacid can also be used, and this can have the effect of promotingsustained duration in the circulation. Such compositions can influencethe physical state, stability, rate of in vivo release, and rate of invivo clearance of the present proteins and derivatives. See, e.g.,Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack PublishingCo., Easton, Pa. 18042) pages 1435-1712, which are herein incorporatedby reference in their entirety. The compositions can be prepared inliquid form, or can be in dried powder, such as lyophilized form.Implantable sustained release formulations are also useful, as aretransdermal or transmucosal formulations. Additionally (oralternatively), the present invention provides compositions for use inany of the various slow or sustained release formulations ormicroparticle formulations known to the skilled artisan, for example,sustained release microparticle formulations, which can be administeredvia pulmonary, intranasal, or subcutaneous delivery routes.

One can dilute the inventive compositions or increase the volume of thepharmaceutical compositions of the invention with an inert material.Such diluents can include carbohydrates, especially, mannitol,α-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans andstarch. Certain inorganic salts may also be used as fillers, includingcalcium triphosphate, magnesium carbonate and sodium chloride. Somecommercially available diluents are Fast-Flo, Emdex, STA-Rx 1500,Emcompress and Avicell.

A variety of conventional thickeners are useful in creams, ointments,suppository and gel configurations of the pharmaceutical composition,such as, but not limited to, alginate, xanthan gum, or petrolatum, mayalso be employed in such configurations of the pharmaceuticalcomposition of the present invention. A permeation or penetrationenhancer, such as polyethylene glycol monolaurate, dimethyl sulfoxide,N-vinyl-2-pyrrolidone, N-(2-hydroxyethyl)-pyrrolidone, or3-hydroxy-N-methyl-2-pyrrolidone can also be employed. Useful techniquesfor producing hydrogel matrices are known. (E.g., Feijen, Biodegradablehydrogel matrices for the controlled release of pharmacologically activeagents, U.S. Pat. No. 4,925,677; Shah et al., BiodegradablepH/thermosensitive hydrogels for sustained delivery of biologicallyactive agents, WO 00/38651 A1). Such biodegradable gel matrices can beformed, for example, by crosslinking a proteinaceous component and apolysaccharide or mucopolysaccharide component, then loading with theinventive composition of matter to be delivered.

Liquid pharmaceutical compositions of the present invention that aresterile solutions or suspensions can be administered to a patient byinjection, for example, intramuscularly, intrathecally, epidurally,intravascularly (e.g., intravenously or intraarterially),intraperitoneally or subcutaneously. (See, e.g., Goldenberg et al.,Suspensions for the sustained release of proteins, U.S. Pat. No.6,245,740 and WO 00/38652 A1). Sterile solutions can also beadministered by intravenous infusion. The inventive composition can beincluded in a sterile solid pharmaceutical composition, such as alyophilized powder, which can be dissolved or suspended at a convenienttime before administration to a patient using sterile water, saline,buffered saline or other appropriate sterile injectable medium.

Implantable sustained release formulations are also useful embodimentsof the inventive pharmaceutical compositions. For example, thepharmaceutically acceptable carrier, being a biodegradable matriximplanted within the body or under the skin of a human or non-humanvertebrate, can be a hydrogel similar to those described above.Alternatively, it may be formed from a poly-alpha-amino acid component.(Sidman, Biodegradable, implantable drug delivery device, and processfor preparing and using same, U.S. Pat. No. 4,351,337). Other techniquesfor making implants for delivery of drugs are also known and useful inaccordance with the present invention.

In powder forms, the pharmaceutically acceptable carrier is a finelydivided solid, which is in admixture with finely divided activeingredient(s), including the inventive composition. For example, in someembodiments, a powder form is useful when the pharmaceutical compositionis configured as an inhalant. (See, e.g., Zeng et al., Method ofpreparing dry powder inhalation compositions, WO 2004/017918; Trunk etal., Salts of the CGRP antagonist BIBN4096 and inhalable powderedmedicaments containing them, U.S. Pat. No. 6,900,317).

One can dilute or increase the volume of the compound of the inventionwith an inert material. These diluents could include carbohydrates,especially mannitol, α-lactose, anhydrous lactose, cellulose, sucrose,modified dextrans and starch. Certain inorganic salts can also be usedas fillers including calcium triphosphate, magnesium carbonate andsodium chloride. Some commercially available diluents are Fast-Flo™,Emdex™, STA-Rx™ 1500, Emcompress™ and Avicell™

Disintegrants can be included in the formulation of the pharmaceuticalcomposition into a solid dosage form. Materials used as disintegrantsinclude but are not limited to starch including the commercialdisintegrant based on starch, Explotab™. Sodium starch glycolate,Amberlite™, sodium carboxymethylcellulose, ultramylopectin, sodiumalginate, gelatin, orange peel, acid carboxymethyl cellulose, naturalsponge and bentonite can all be used. Insoluble cationic exchange resinis another form of disintegrant. Powdered gums can be used asdisintegrants and as binders and these can include powdered gums such asagar, Karaya or tragacanth. Alginic acid and its sodium salt are alsouseful as disintegrants.

Binders can be used to hold the therapeutic agent together to form ahard tablet and include materials from natural products such as acacia,tragacanth, starch and gelatin. Others include methyl cellulose (MC),ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both beused in alcoholic solutions to granulate the therapeutic.

An antifrictional agent can be included in the formulation of thetherapeutic to prevent sticking during the formulation process.Lubricants can be used as a layer between the therapeutic and the diewall, and these can include but are not limited to; stearic acidincluding its magnesium and calcium salts, polytetrafluoroethylene(PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricantscan also be used such as sodium lauryl sulfate, magnesium laurylsulfate, polyethylene glycol of various molecular weights, Carbowax 4000and 6000.

Glidants that might improve the flow properties of the drug duringformulation and to aid rearrangement during compression might be added.The glidants can include starch, talc, pyrogenic silica and hydratedsilicoaluminate.

To aid dissolution of the compound of this invention into the aqueousenvironment a surfactant might be added as a wetting agent. Surfactantscan include anionic detergents such as sodium lauryl sulfate, dioctylsodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergentsmight be used and could include benzalkonium chloride or benzethoniumchloride. The list of potential nonionic detergents that could beincluded in the formulation as surfactants are lauromacrogol 400,polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fattyacid ester, methyl cellulose and carboxymethyl cellulose. Thesesurfactants could be present in the formulation of the protein orderivative either alone or as a mixture in different ratios.

Oral Dosage Forms.

Also useful are oral dosage forms of the inventive compositionss. Ifnecessary, the composition can be chemically modified so that oraldelivery is efficacious. Generally, the chemical modificationcontemplated is the attachment of at least one moiety to the moleculeitself, where said moiety permits (a) inhibition of proteolysis; and (b)uptake into the blood stream from the stomach or intestine. Also desiredis the increase in overall stability of the compound and increase incirculation time in the body. Moieties useful as covalently attachedhalf-life extending moieties in this invention can also be used for thispurpose. Examples of such moieties include: PEG, copolymers of ethyleneglycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinylalcohol, polyvinyl pyrrolidone and polyproline. See, for example,Abuchowski and Davis (1981), Soluble Polymer-Enzyme Adducts, Enzymes asDrugs (Hocenberg and Roberts, eds.), Wiley-Interscience, New York, N.Y.,pp 367-83; Newmark, et al. (1982), J. Appl. Biochem. 4:185-9. Otherpolymers that could be used are poly-1,3-dioxolane andpoly-1,3,6-tioxocane. Preferred for pharmaceutical usage, as indicatedabove, are PEG moieties.

For oral delivery dosage forms, it is also possible to use a salt of amodified aliphatic amino acid, such as sodiumN-(8-[2-hydroxybenzoyl]amino) caprylate (SNAC), as a carrier to enhanceabsorption of the therapeutic compounds of this invention. The clinicalefficacy of a heparin formulation using SNAC has been demonstrated in aPhase II trial conducted by Emisphere Technologies. See U.S. Pat. No.5,792,451, “Oral drug delivery composition and methods.”

In one embodiment, the pharmaceutically acceptable carrier can be aliquid and the pharmaceutical composition is prepared in the form of asolution, suspension, emulsion, syrup, elixir or pressurizedcomposition. The active ingredient(s) (e.g., the inventive compositionof matter) can be dissolved, diluted or suspended in a pharmaceuticallyacceptable liquid carrier such as water, an organic solvent, a mixtureof both, or pharmaceutically acceptable oils or fats. The liquid carriercan contain other suitable pharmaceutical additives such as detergentsand/or solubilizers (e.g., Tween 80, Polysorbate 80), emulsifiers,buffers at appropriate pH (e.g., Tris-HCl, acetate, phosphate),adjuvants, anti-oxidants (e.g., ascorbic acid, sodium metabisulfite),preservatives (e.g., Thimersol, benzyl alcohol), sweeteners, flavoringagents, suspending agents, thickening agents, bulking substances (e.g.,lactose, mannitol), colors, viscosity regulators, stabilizers,electrolytes, osmolutes or osmo-regulators. Additives can also beincluded in the formulation to enhance uptake of the inventivecomposition. Additives potentially having this property are for instancethe fatty acids oleic acid, linoleic acid and linolenic acid.

Useful are oral solid dosage forms, which are described generally inRemington's Pharmaceutical Sciences (1990), supra, in Chapter 89, whichis hereby incorporated by reference in its entirety. Solid dosage formsinclude tablets, capsules, pills, troches or lozenges, cachets orpellets. Also, liposomal or proteinoid encapsulation can be used toformulate the present compositions (as, for example, proteinoidmicrospheres reported in U.S. Pat. No. 4,925,673). Liposomalencapsulation can be used and the liposomes can be derivatized withvarious polymers (e.g., U.S. Pat. No. 5,013,556). A description ofpossible solid dosage forms for the therapeutic is given in Marshall,K., Modern Pharmaceutics (1979), edited by G. S. Banker and C. T.Rhodes, in Chapter 10, which is hereby incorporated by reference in itsentirety. In general, the formulation will include the inventivecompound, and inert ingredients that allow for protection against thestomach environment, and release of the biologically active material inthe intestine.

The composition of this invention can be included in the formulation asfine multiparticulates in the form of granules or pellets of particlesize about 1 mm. The formulation of the material for capsuleadministration could also be as a powder, lightly compressed plugs oreven as tablets. The therapeutic could be prepared by compression.

Colorants and flavoring agents can all be included. For example, theprotein (or derivative) can be formulated (such as by liposome ormicrosphere encapsulation) and then further contained within an edibleproduct, such as a refrigerated beverage containing colorants andflavoring agents.

In tablet form, the active ingredient(s) are mixed with apharmaceutically acceptable carrier having the necessary compressionproperties in suitable proportions and compacted in the shape and sizedesired.

The powders and tablets preferably contain up to 99% of the activeingredient(s). Suitable solid carriers include, for example, calciumphosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch,gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ionexchange resins.

Controlled release formulation can be desirable. The composition of thisinvention could be incorporated into an inert matrix that permitsrelease by either diffusion or leaching mechanisms e.g., gums. Slowlydegenerating matrices can also be incorporated into the formulation,e.g., alginates, polysaccharides. Another form of a controlled releaseof the compositions of this invention is by a method based on the Oros™therapeutic system (Alza Corp.), i.e., the drug is enclosed in asemipermeable membrane which allows water to enter and push drug outthrough a single small opening due to osmotic effects. Some entericcoatings also have a delayed release effect.

Other coatings can be used for the formulation. These include a varietyof sugars that could be applied in a coating pan. The therapeutic agentcould also be given in a film-coated tablet and the materials used inthis instance are divided into 2 groups. The first are the nonentericmaterials and include methylcellulose, ethyl cellulose, hydroxyethylcellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose,hydroxypropyl-methyl cellulose, sodium carboxymethyl cellulose,providone and the polyethylene glycols. The second group consists of theenteric materials that are commonly esters of phthalic acid.

A mix of materials might be used to provide the optimum film coating.Film coating can be carried out in a pan coater or in a fluidized bed orby compression coating.

Pulmonary Delivery Forms.

Pulmonary delivery of the inventive compositions is also useful. Theprotein (or derivative) is delivered to the lungs of a mammal whileinhaling and traverses across the lung epithelial lining to the bloodstream. (Other reports of this include Adjei et al., Pharma. Res. (1990)7: 565-9; Adjei et al. (1990), Internatl. J. Pharmaceutics 63: 135-44(leuprolide acetate); Braquet et al. (1989), J. Cardiovasc. Pharmacol.13 (suppl. 5): s.143-146 (endothelin-1); Hubbard et al. (1989), AnnalsInt. Med. 3: 206-12 (al-antitrypsin); Smith et al. (1989), J. Clin.Invest. 84: 1145-6 (al-proteinase); Oswein et al. (March 1990),“Aerosolization of Proteins,” Proc. Symp. Resp. Drug Delivery II,Keystone, Colo. (recombinant human growth hormone); Debs et al. (1988),J. Immunol. 140: 3482-8 (interferon-α and tumor necrosis factor α) andPlatz et al., U.S. Pat. No. 5,284,656 (granulocyte colony stimulatingfactor). Useful in the practice of this invention are a wide range ofmechanical devices designed for pulmonary delivery of therapeuticproducts, including but not limited to nebulizers, metered doseinhalers, and powder inhalers, all of which are familiar to thoseskilled in the art. Some specific examples of commercially availabledevices suitable for the practice of this invention are the Ultraventnebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the AcornII nebulizer, manufactured by Marquest Medical Products, Englewood,Colo.; the Ventolin metered dose inhaler, manufactured by Glaxo Inc.,Research Triangle Park, N.C.; and the Spinhaler powder inhaler,manufactured by Fisons Corp., Bedford, Mass. (See, e.g., Helgesson etal., Inhalation device, U.S. Pat. No. 6,892,728; McDerment et al., Drypowder inhaler, WO 02/11801 A1; Ohki et al., Inhalant medicator, U.S.Pat. No. 6,273,086).

All such devices require the use of formulations suitable for thedispensing of the inventive compound. Typically, each formulation isspecific to the type of device employed and can involve the use of anappropriate propellant material, in addition to diluents, adjuvantsand/or carriers useful in therapy.

The inventive compound should most advantageously be prepared inparticulate form with an average particle size of less than 10 μm (ormicrons), most preferably 0.5 to 5 μm, for most effective delivery tothe distal lung.

Pharmaceutically acceptable carriers include carbohydrates such astrehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Otheringredients for use in formulations can include DPPC, DOPE, DSPC andDOPC. Natural or synthetic surfactants can be used. PEG can be used(even apart from its use in derivatizing the protein or analog).Dextrans, such as cyclodextran, can be used. Bile salts and otherrelated enhancers can be used. Cellulose and cellulose derivatives canbe used Amino acids can be used, such as use in a buffer formulation.

Also, the use of liposomes, microcapsules or microspheres, inclusioncomplexes, or other types of carriers is contemplated.

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise the inventive compound dissolved inwater at a concentration of about 0.1 to 25 mg of biologically activeprotein per mL of solution. The formulation can also include a bufferand a simple sugar (e.g., for protein stabilization and regulation ofosmotic pressure). The nebulizer formulation can also contain asurfactant, to reduce or prevent surface induced aggregation of theprotein caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing the inventive compoundsuspended in a propellant with the aid of a surfactant. The propellantcan be any conventional material employed for this purpose, such as achlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid can also be useful as a surfactant. (See,e.g., Backstrom et al., Aerosol drug formulations containinghydrofluoroalkanes and alkyl saccharides, U.S. Pat. No. 6,932,962).

Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing the inventive compound and can alsoinclude a bulking agent, such as lactose, sorbitol, sucrose, mannitol,trehalose, or xylitol in amounts which facilitate dispersal of thepowder from the device, e.g., 50 to 90% by weight of the formulation.

Nasal Delivery Forms.

In accordance with the present invention, intranasal delivery of theinventive composition of matter and/or pharmaceutical compositions isalso useful, which allows passage thereof to the blood stream directlyafter administration to the inside of the nose, without the necessityfor deposition of the product in the lung. Formulations suitable forintransal administration include those with dextran or cyclodextran, andintranasal delivery devices are known. (See, e.g, Freezer, Inhaler, U.S.Pat. No. 4,083,368).

Transdermal and Transmucosal

(e.g., buccal) delivery forms). In some embodiments, the inventivecomposition is configured as a part of a pharmaceutically acceptabletransdermal or transmucosal patch or a troche. Transdermal patch drugdelivery systems, for example, matrix type transdermal patches, areknown and useful for practicing some embodiments of the presentpharmaceutical compositions. (E.g., Chien et al., Transdermalestrogen/progestin dosage unit, system and process, U.S. Pat. Nos.4,906,169 and 5,023,084; Cleary et al., Diffusion matrix for transdermaldrug administration and transdermal drug delivery devices includingsame, U.S. Pat. No. 4,911,916; Teillaud et al., EVA-based transdermalmatrix system for the administration of an estrogen and/or aprogestogen, U.S. Pat. No. 5,605,702; Venkateshwaran et al., Transdermaldrug delivery matrix for coadministering estradiol and another steroid,U.S. Pat. No. 5,783,208; Ebert et al., Methods for providingtestosterone and optionally estrogen replacement therapy to women, U.S.Pat. No. 5,460,820). A variety of pharmaceutically acceptable systemsfor transmucosal delivery of therapeutic agents are also known in theart and are compatible with the practice of the present invention.(E.g., Heiber et al., Transmucosal delivery of macromolecular drugs,U.S. Pat. Nos. 5,346,701 and 5,516,523; Longenecker et al.,Transmembrane formulations for drug administration, U.S. Pat. No.4,994,439).

Buccal delivery of the inventive compositions is also useful. Buccaldelivery formulations are known in the art for use with peptides. Forexample, known tablet or patch systems configured for drug deliverythrough the oral mucosa (e.g., sublingual mucosa), include someembodiments that comprise an inner layer containing the drug, apermeation enhancer, such as a bile salt or fusidate, and a hydrophilicpolymer, such as hydroxypropyl cellulose, hydroxypropyl methylcellulose,hydroxyethyl cellulose, dextran, pectin, polyvinyl pyrrolidone, starch,gelatin, or any number of other polymers known to be useful for thispurpose. This inner layer can have one surface adapted to contact andadhere to the moist mucosal tissue of the oral cavity and can have anopposing surface adhering to an overlying non-adhesive inert layer.Optionally, such a transmucosal delivery system can be in the form of abilayer tablet, in which the inner layer also contains additionalbinding agents, flavoring agents, or fillers. Some useful systems employa non-ionic detergent along with a permeation enhancer. Transmucosaldelivery devices may be in free form, such as a cream, gel, or ointment,or may comprise a determinate form such as a tablet, patch or troche.For example, delivery of the inventive composition can be via atransmucosal delivery system comprising a laminated composite of, forexample, an adhesive layer, a backing layer, a permeable membranedefining a reservoir containing the inventive composition, a peel sealdisc underlying the membrane, one or more heat seals, and a removablerelease liner. (E.g., Ebert et al., Transdermal delivery system withadhesive overlay and peel seal disc, U.S. Pat. No. 5,662,925; Chang etal., Device for administering an active agent to the skin or mucosa,U.S. Pat. Nos. 4,849,224 and 4,983,395). These examples are merelyillustrative of available transmucosal drug delivery technology and arenot limiting of the present invention.

Dosages.

The dosage regimen involved in a method for treating the above-describedconditions will be determined by the attending physician, consideringvarious factors which modify the action of drugs, e.g. the age,condition, body weight, sex and diet of the patient, the severity of anyinfection, time of administration and other clinical factors. Generally,the daily regimen should be in the range of 0.1-1000 micrograms of theinventive compound per kilogram of body weight, preferably 0.1-150micrograms per kilogram.

The following working examples are illustrative and not to be construedin any way as limiting the scope of the present invention.

EXAMPLES Example 1 huFc(IgG1) Variants Constructed for BacterialExpression

By way of example, four variants of human Fc domain (huFc(IgG1) wereconstructed.

-   -   1) huFc(IgG1) with a Q143C mutation (at position 143 of SEQ ID        NO:599; designated “Strain 13300”) was made as follows. Two PCR        fragments introducing the Q143C mutation were amplified from a        plasmid encoding the huFc(IgG1). The first PCR fragment was        amplified by the following two primers:    -   3430-37    -   GAGGAATAACATATGGACAAAACTCACACATGTCCACCT (SEQ ID NO: 641), which        encodes the first 8 amino acids of huFc(IgG1) plus a        15-nucleotide 5′ extension including a NdeI site; and    -   4220-28    -   GGTCAGGCTGACGCAGTTCTTGGTCAG (SEQ ID NO: 642), which encodes 9        amino acids of huFc(IgG1) from the 139th to the 147th amino acid        with the 143th amino acid mutated from Glutamine to Cysteine in        the 3′ orientation.    -   The second PCR fragment was amplified by the following two        primers:    -   4220-27    -   CTGACCAAGAACTGCGTCAGCCTGACC (SEQ ID NO: 643), which encodes 9        amino acids of huFc(IgG1) from the 139th to the 147th amino acid        with the 143th amino acid mutated from Glutamine to Cysteine in        the 5′ orientation.    -   3421-87    -   CCGCGGCGTCTCGAGATTATTTACCCGGAGACAGGGAGAGGCT (SEQ ID NO: 644),        which encodes the last 8 amino acids of huFc(IgG1), a stop codon        and a 15-nucleotide 3′ extension including a XhoI site.    -   The 2 PCR fragments were again amplified with primers 3430-37        and 3421-87. The PCR product was cloned in pAMG21 vector and        sequence-confirmed by DNA sequencing. The E. coli strain that        harbors this plasmid is named strain 13300.    -   The relevant coding sequence of Strain 13300 is:

SEQ ID NO: 645 1 ATGGACAAAA CTCACACATG TCCACCTTGC CCAGCACCTG AACTCCTGGG51 GGGACCGTCA GTTTTCCTCT TCCCCCCAAA ACCCAAGGAC ACCCTCATGA 101 TCTCCCGGACCCCTGAGGTC ACATGCGTGG TGGTGGACGT GAGCCACGAA 151 GACCCTGAGG TCAAGTTCAACTGGTACGTG GACGGCGTGG AGGTGCATAA 201 TGCCAAGACA AAGCCGCGGG AGGAGCAGTACAACAGCACG TACCGTGTGG 251 TCAGCGTCCT CACCGTCCTG CACCAGGACT GGCTGAATGGCAAGGAGTAC 301 AAGTGCAAGG TCTCCAACAA AGCCCTCCCA GCCCCCATCG AGAAAACCAT351 CTCCAAAGCC AAAGGGCAGC CCCGAGAACC ACAGGTGTAC ACCCTGCCCC 401CATCCCGGGA TGAGCTGACC AAGAACTGCG TCAGCCTGAC CTGCCTGGTC 451 AAAGGCTTCTATCCCAGCGA CATCGCCGTG GAGTGGGAGA GCAATGGGCA 501 GCCGGAGAAC AACTACAAGACCACGCCTCC CGTGCTGGAC TCCGACGGCT 551 CCTTCTTCCT CTACAGCAAG CTCACCGTGGACAAGAGCAG GTGGCAGCAG 601 GGGAACGTCT TCTCATGCTC CGTGATGCAT GAGGCTCTGCACAACCACTA 651 CACGCAGAAG AGCCTCTCCC TGTCTCCGGG TAAATAAT//

-   -   The translation of this nucleotide sequence is as follows:    -   Strain 13300 (huFC(IgG1)Q143C):

(SEQ ID NO: 646) 1 MDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEVTCVVVDVSHE 51 DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY 101KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNCVSLTCLV 151 KGFYPSDIAVEWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ 201 GNVFSCSVMH EALHNHYTQKSLSLSPGK//.

-   -   2) huFc(IgG1) with a L139C mutation (at position 139 of SEQ ID        NO:599; designated “Strain 13322”) was made following procedures        similar to those described in (1) above using the following        primers and procedures.    -   The first PCR fragment was amplified by the following two        primers:    -   3430-37    -   GAGGAATAACATATGGACAAAACTCACACATGTCCACCT (SEQ ID NO: 641), which        encodes the first 8 amino acids of huFc(IgG1) plus a        15-nucleotide 5′ extension including a NdeI site and    -   4220-26    -   CTGGTTCTTGGTGCACTCATCCCGGGA (SEQ ID NO: 647), which encodes 9        amino acids of huFc(IgG1) from the 135th to the 143th amino acid        with the 138th amino acid mutated from Leucine to Cysteine in        the 3′ orientation.    -   The second PCR fragment was amplified by the following two        primers:    -   4220-25    -   TCCCGGGATGAGTGCACCAAGAACCAG (SEQ ID NO: 648), which encodes 9        amino acids of huFc(IgG1) from the 135th to the 143th amino acid        with the 138th amino acid mutated from Leucine to Cysteine in        the 5′ orientation.    -   3421-87    -   CCGCGGCGTCTCGAGATTATTTACCCGGAGACAGGGAGAGGCT (SEQ ID NO: 644),        which encodes the last 8 amino acids of huFc(IgG1), a stop codon        and a 15-nucleotide 3′ extension including a XhoI site. The 2        PCR fragments were again amplified with primers 3430-37 and        3421-87. The PCR product was cloned in pAMG21 vector and        sequenced-confirmed by DNA sequencing. The E. coli strain that        harbors this plasmid is named strain 13322.    -   The relevant coding sequence of Strain 13322 is:

SEQ ID NO: 649 1 ATGGACAAAA CTCACACATG TCCACCTTGC CCAGCACCTG AACTCCTGGG51 GGGACCGTCA GTTTTCCTCT TCCCCCCAAA ACCCAAGGAC ACCCTCATGA 101 TCTCCCGGACCCCTGAGGTC ACATGCGTGG TGGTGGACGT GAGCCACGAA 151 GACCCTGAGG TCAAGTTTAACTGGTACGTG GACGGCGTGG AGGTGCATAA 201 TGCCAAGACA AAGCCGCGGG AGGAGCAGTACAACAGCACG TACCGTGTGG 251 TCAGCGTCCT CACCGTCCTG CACCAGGACT GGCTGAATGGCAAGGAGTAC 301 AAGTGCAAGG TCTCCAACAA AGCCCTCCCA GCCCCCATCG AGAAAACCAT351 CTCCAAAGCC AAAGGGCAGC CCCGAGAACC ACAGGTGTAC ACCCTGCCCC 401CATCCCGGGA TGAGTGCACC AAGAACCAGG TCAGCCTGAC CTGCCTGGTC 451 AAAGGCTTCTATCCCAGCGA CATCGCCGTG GAGTGGGAGA GCAATGGGCA 501 GCCGGAGAAC AACTACAAGACCACGCCTCC CGTGCTGGAC TCCGACGGCT 551 CCTTCTTCCT CTACAGCAAG CTCACCGTGGACAAGAGCAG GTGGCAGCAG 601 GGGAACGTCT TCTCATGCTC CGTGATGCAT GAGGCTCTGCACAACCACTA 651 CACGCAGAAG AGCCTCTCCC TGTCTCCGGG TAAATAAT//.

-   -   The translation of this nucleotide sequence is as follows:    -   Strain 13322 (huFc(IgG1)L139C):

(SEQ ID NO: 650) 1 MDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEVTCVVVDVSHE 51 DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY 101KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDECT KNQVSLTCLV 151 KGFYPSDIAVEWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ 201 GNVFSCSVMH EALHNHYTQKSLSLSPGK//.

-   -   3) huFc(IgG1) with a S145C mutation (at position 145 of SEQ ID        NO:599; designated “Strain 13323”) was made following procedures        similar to those described in (1) above using the following        primers and procedures. The first PCR fragment was amplified by        the following two primers:    -   3430-37    -   GAGGAATAACATATGGACAAAACTCACACATGTCCACCT (SEQ ID NO: 641) which        encodes the first 8 amino acids of huFc(IgG1) plus a        15-nucleotide 5′ extension including a NdeI site and    -   4220-30    -   CAGGCAGGTCAGGCAGACCTGGTTCTT (SEQ ID NO: 651), which encodes the        9 amino acids of huFc(IgG1) from the 141th to the 149th amino        acid with the 145th amino acid mutated from Serine to Cysteine        in the 3′ orientation.    -   The second PCR fragment was amplified by the following two        primers:    -   4220-29    -   AAGAACCAGGTCTGCCTGACCTGCCTG (SEQ ID NO: 652), which encodes the        9 amino acids of huFc(IgG1) from the 141th to the 149th amino        acid with the 145th amino acid mutated from Serine to Cysteine        in the 5′ orientation.    -   3421-87    -   CCGCGGCGTCTCGAGATTATTTACCCGGAGACAGGGAGAGGCT (SEQ ID NO: 644),        which encodes the last 8 amino acids of huFc(IgG1), a stop codon        and a 15-nucleotide 3′ extension including a XhoI site. The 2        PCR fragments were again amplified with primers 3430-37 and        3421-87. The PCR product was cloned in pAMG21 vector and        sequence-confirmed by DNA sequencing. The E. coli strain that        harbors this plasmid is named strain 13323.    -   The relevant coding sequence of Strain 13323 is:

SEQ ID NO: 653 1 ATGGACAAAA CTCACACATG TCCACCTTGC CCAGCACCTG AACTCCTGGG51 GGGACCGTCA GTTTTCCTCT TCCCCCCAAA ACCCAAGGAC ACCCTCATGA 101 TCTCCCGGACCCCTGAGGTC ACATGCGTGG TGGTGGACGT GAGCCACGAA 151 GACCCTGAGG TCAAGTTCAACTGGTACGTG GACGGCGTGG AGGTGCATAA 201 TGCCAAGACA AAGCCGCGGG AGGAGCAGTACAACAGCACG TACCGTGTGG 251 TCAGCGTCCT CACCGTCCTG CACCAGGACT GGCTGAATGGCAAGGAGTAC 301 AAGTGCAAGG TCTCCAACAA AGCCCTCCCA GCCCCCATCG AGAAAACCAT351 CTCCAAAGCC AAAGGGCAGC CCCGAGAACC ACAGGTGTAC ACCCTGCCCC 401CATCCCGGGA TGAGCTGACC AAGAACCAGG TCTGCCTGAC CTGCCTGGTC 451 AAAGGCTTCTATCCCAGCGA CATCGCCGTG GAGTGGGAGA GCAATGGGCA 501 GCCGGAGAAC AACTACAAGACCACGCCTCC CGTGCTGGAC TCCGACGGCT 551 CCTTCTTCCT CTACAGCAAG CTCACCGTGGACAAGAGCAG GTGGCAGCAG 601 GGGAACGTCT TCTCATGCTC CGTGATGCAT GAGGCTCTGCACAACCACTA 651 CACGCAGAAG AGCCTCTCCC TGTCTCCGGG TAAATAAT//

-   -   The translation of this sequence is as follows:    -   Strain 13323 (huFc(IgG1)S145C):

(SEQ ID NO: 654) 1 MDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEVTCVVVDVSHE 51 DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY 101KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVCLTCLV 151 KGFYPSDIAVEWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ 201 GNVFSCSVMH EALHNHYTQKSLSLSPGK//.

-   -   4) huFc(IgG1) with a S196C mutation (at position 196 of SEQ ID        NO:599; designated “Strain 13324”) was made following procedures        similar to those described in (1) above using the following        primers and procedures. The first PCR fragment was amplified by        the following two primers:    -   3430-37    -   GAGGAATAACATATGGACAAAACTCACACATGTCCACCT (SEQ ID NO: 641), which        encodes the first 8 amino acids of huFc(IgG1) plus a        15-nucleotide 5′ extension including a NdeI site and    -   4220-32    -   CTGCTGCCACCTGCACTTGTCCACGGT (SEQ ID NO: 655), which encodes 9        amino acids of huFc(IgG1) from the 192th to the 200th amino acid        with the 196th amino acid mutated from Serine to Cysteine in the        3′ orientation.    -   The second PCR fragment was amplified by the following two        primers:    -   4220-31    -   ACCGTGGACAAGTGCAGGTGGCAGCAG (SEQ ID NO: 656), which encodes 9        amino acids of huFc(IgG1) from the 192th to the 200th amino acid        with the 196th amino acid mutated from Serine to Cysteine in the        5′ orientation.    -   3421-87    -   CCGCGGCGTCTCGAGATTATTTACCCGGAGACAGGGAGAGGCT (SEQ ID NO: 644),        which encodes the last 8 amino acids of huFc(IgG1), a stop codon        and a 15-nucleotide 3′ extension including a XhoI site. The 2        PCR fragments were again amplified with primers 3430-37 and        3421-87. The PCR product was cloned in pAMG21 vector and        sequenced-confirmed by DNA sequencing. The E. coli strain that        harbors this plasmid is named strain 13324.    -   The relevant coding sequence of Strain 13324 is:

SEQ ID NO: 657 1 ATGGACAAAA CTCACACATG TCCACCTTGC CCAGCACCTG AACTCCTGGG51 GGGACCGTCA GTTTTCCTCT TCCCCCCAAA ACCCAAGGAC ACCCTCATGA 101 TCTCCCGGACCCCTGAGGTC ACATGCGTGG TGGTGGACGT GAGCCACGAA 151 GACCCTGAGG TCAAGTTCAACTGGTACGTG GACGGCGTGG AGGTGCATAA 201 TGCCAAGACA AAGCCGCGGG AGGAGCAGTACAACAGCACG TACCGTGTGG 251 TCAGCGTCCT CACCGTCCTG CACCAGGACT GGCTGAATGGCAAGGAGTAC 301 AAGTGCAAGG TCTCCAACAA AGCCCTCCCA GCCCCCATCG AGAAAACCAT351 CTCCAAAGCC AAAGGGCAGC CCCGAGAACC ACAGGTGTAC ACCCTGCCCC 401CATCCCGGGA TGAGCTGACC AAGAACCAGG TCAGCCTGAC CTGCCTGGTC 451 AAAGGCTTCTATCCCAGCGA CATCGCCGTG GAGTGGGAGA GCAATGGGCA 501 GCCGGAGAAC AACTACAAGACCACGCCTCC CGTGCTGGAC TCCGACGGCT 551 CCTTCTTCCT CTACAGCAAG CTCACCGTGGACAAGTGCAG GTGGCAGCAG 601 GGGAACGTCT TCTCATGCTC CGTGATGCAT GAGGCTCTGCACAACCACTA 651 CACGCAGAAG AGCCTCTCCC TGTCTCCGGG TAAATAAT//

-   -   The translation of this nucleotide sequence is as follows:    -   Strain 13324 (huFC(IgG1)S196C):

(SEQ ID NO: 658) 1 MDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEVTCVVVDVSHE 51 DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY 101KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV 151 KGFYPSDIAVEWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKCRWQQ 201 GNVFSCSVMH EALHNHYTQKSLSLSPGK//.

Example 2 Purification of Fc-Cysteine Analogs

The cDNA clones constructed as described in Example 1 hereinabove:Strain 13300 (huFc(IgG1)Q143C), Strain 13322 (huFc(IgG1)L139C), Strain13323 (huFc(IgG1)S145C) and Strain 13324 (huFc(IgG1)S196C) weretransformed in E. coli and expressed in inclusion bodies. Cell pastesfrom each strain were resuspended in 10 ml water/g paste, lysed by threepassages through a microfluidizer and the insoluble inclusion body (IB)fraction was collected by centrifugation. The IBs were subsequentlywashed with 1% deoxycholate, centrifuged and washed again with water.The final pellet of deoxycholate washed inclusion bodies (DWIBs) wasweighed and frozen at −80° C.

The frozen DWIBs, from each clone, were then thawed and resuspended in 1ml water/g DWIBs, then solubilized and reduced by the addition of 9 mlof 8 M buffered Gdn-HCl, 11 mM DTT. The solubilization proceeded at roomtemperature, with stirring, for 1 hour.

Each huFc-cysteine analog was then refolded by rapid dilution (20-fold)of the solubilized DWIBs into a refolding buffer consisting of 2 M Urea,150 mM Arginine, 50 mM Tris, 1 mM Cysteine, 3 mM Cystamine, pH 8.5. Therefolding reaction was allowed to proceed for 48-72 hours with gentlestirring at 4° C.

Purification of the huFc-cysteine analogs began affinity columnchromatography using MAb Select resin (GE Healthcare, Piscataway, N.J.).Briefly, the refold reaction was clarified by centrifugation followed by0.45 micron filtration. The filtered refold was then loaded to a MAbselect column pre-equilibrated in PBS. After loading, the column wasfurther washed with 3 column volumes PBS, then eluted with 0.1 N aceticacid. The protein fraction that was acid eluted from the MAb Selectcolumn was immediately dialyzed into 10 mM NaOAc, 50 mM NaCl, pH 5.0.

Additional purification was achieved by cation exchange chromatographyusing SP Sepharose HP resin (GE Healthcare, Piscataway, N.J.). Briefly,after dialysis, the MAb Select eluted pool was loaded to the ionexchange column, pre-equilibrated in 10 mM NaOAc, 50 mM NaCl, pH 5.0,washed with 3 column volumes of equilibration buffer, then eluted with alinear 50-500 mM NaCl gradient. The eluted peaks were evaluated bySDS-PAGE and the peaks containing protein of approximately 51 kD werepooled and concentrated. The concentrated pools were then dialyzed intoPBS and concentrations determined spectrascopically using calculatedextinction coefficients.

The final pools were analyzed by SDS-PAGE, SEC-HPLC, RP-HPLC and LC-MS.FIG. 5 shows the purity by SDS-PAGE gel of the 4 purified huFc-cysteineanalogs. FIG. 6 shows the purity of clone 13324 huFCS196C by SEC-HPLC.FIG. 7 shows the purity and mass determinations of clone 13324huFc(S196C) by LC-MS. Additional mass observed is consistent withcystamine adducts carried over from the refold reaction. The observedmass differential disappears when the samples are reduced prior to LC-MSanalyses, further indicating a mixed disulphide adduct is present.

Example 3 Conjugation of Polyethylene Glycol (PEG) to Fc-CysteineAnalogs

The huFc (S196C) analog, clone #13324, was selected as representative ofthe huFc-cysteine analogs produced, as described in Example 2 hereinabove, for the purpose of developing a site-selective conjugationprocess for the Fc-cysteine analogs. Since the LC-MS analyses of thepurified huFc-cysteine analogs indicated the presence of mixeddisulphides with low molecular weight adducts, a limited reduction stepwas undertaken prior to conjugation. This was followed by thiol specificPEGylation to assess the degree and site-selectivity of conjugation atthe engineered cysteine.

Briefly, the huFc (S196C) analog described in Example 2 was partiallyreduced by titrating ris(2-carboxyethylphosphine) hydrochloride (TCEP)from 0-5 molar excess stoichiometries relative to the concentration ofengineered cysteine. The reduction reaction was incubated 2 hours, atroom temperature, in 50 mM sodium phosphate, 5 mM EDTA, pH 6.0 at aprotein concentration of 1 mg/ml. TCEP and reduced adduct were removedby gel filtration using disposable Zebra desalt spin columns (Pierce,Rockford, Ill.) equilibrated in 50 mM sodium phosphate, 5 mM EDTA, pH6.0. The reduced protein eluted from the gel filtration was then reactedwith 20 kD mPEG-maleimide (Nektar Inc., Huntsville, Ala.) in a 2-foldmolar excess over the engineered cysteine concentration. The PEGylationreaction was allowed to proceed overnight, at room temperature. Theextent of modification was determined by SDS-PAGE (FIG. 8A and FIG. 8B)and by SEC-HPLC (FIG. 9A-B).

Next, the PEGylation reaction was scaled up using a 1:1.25 molar ratioof engineered cysteine to TCEP and the PEG-huFc(S196C) analog waspurified by cation exchange chromatography. Purification was achievedwith an SP Sepharose HP column (GE Healthcare, Piscataway, N.J.)equilibrated in 20 mM sodium acetate, pH 4.0 and was eluted with alinear 0-0.5 M sodium chloride gradient. The eluted peaks were evaluatedby SDS-PAGE and SEC-LS and were pooled and concentrated based on size.FIG. 9A-B shows the SEC-LS result identifying the isolatedPEG-huFc(S196C) conjugate, demonstrating a mass consistent with two 20kD PEG molecules conjugated to one Fc dimer (˜53 kD). Subsequent peptidemapping confirmed Cys 196 as the site of PEGylation in this pool.

ABBREVIATIONS

Abbreviations used throughout this specification are as defined below,unless otherwise defined in specific circumstances.

-   -   Ac acetyl (used to refer to acetylated residues)    -   AcBpa acetylated p-benzoyl-L-phenylalanine    -   ADCC antibody-dependent cellular cytotoxicity    -   Aib aminoisobutyric acid    -   bA beta-alanine    -   Bpa p-benzoyl-L-phenylalanine    -   BrAc bromoacetyl (BrCH₂C(O)    -   BSA Bovine serum albumin    -   Bzl Benzyl    -   Cap Caproic acid    -   CTL Cytotoxic T lymphocytes    -   CTLA4 Cytotoxic T lymphocyte antigen 4    -   DARC Duffy blood group antigen receptor    -   DCC Dicylcohexylcarbodiimide    -   Dde 1-(4,4-dimethyl-2,6-dioxo-cyclohexylidene)ethyl    -   EDTA ethylene diamine tetraacetic acid    -   EMP Erythropoietin-mimetic peptide    -   ESI-MS Electron spray ionization mass spectrometry    -   EPO Erythropoietin    -   Fmoc fluorenylmethoxycarbonyl    -   G-CSF Granulocyte colony stimulating factor    -   GH Growth hormone    -   HCT hematocrit    -   HGB hemoglobin    -   hGH Human growth hormone    -   HOBt 1-Hydroxybenzotriazole    -   HPLC high performance liquid chromatography    -   IL interleukin    -   IL-R interleukin receptor    -   IL-1R interleukin-1 receptor    -   IL-1ra interleukin-1 receptor antagonist    -   Lau Laurie acid    -   LPS lipopolysaccharide    -   LYMPH lymphocytes    -   MALDI-MS Matrix-assisted laser desorption ionization mass        spectrometry    -   Me methyl    -   MeO methoxy    -   MHC major histocompatibility complex    -   MMP matrix metalloproteinase    -   MMPI matrix metalloproteinase inhibitor    -   1-Nap 1-napthylalanine    -   NEUT neutrophils    -   NGF nerve growth factor    -   Nle norleucine    -   NMP N-methyl-2-pyrrolidinone    -   PAGE polyacrylamide gel electrophoresis    -   PBS Phosphate-buffered saline    -   Pbf 2,2,4,6,7-pendamethyldihydrobenzofuran-5-sulfonyl    -   PCR polymerase chain reaction    -   Pec pipecolic acid    -   PEG Poly(ethylene glycol)    -   pGlu pyroglutamic acid    -   Pic picolinic acid    -   PLT platelets    -   pY phosphotyrosine    -   PTFE polytetrafluoroethylene    -   RBC red blood cells    -   RBS ribosome binding site    -   RP-HPLC reversed phase HPLC    -   RT room temperature (25° C.)    -   Sar sarcosine    -   SDS sodium dodecyl sulfate    -   STK serine-threonine kinases    -   t-Boc tert-Butoxycarbonyl    -   tBu tert-Butyl    -   TGF tissue growth factor    -   THF thymic humoral factor    -   TK tyrosine kinase    -   TMP Thrombopoietin-mimetic peptide    -   TNF Tissue necrosis factor    -   TPO Thrombopoietin    -   TRAIL TNF-related apoptosis-inducing ligand    -   Trt trityl    -   UK urokinase    -   UKR urokinase receptor    -   VEGF vascular endothelial cell growth factor    -   VIP vasoactive intestinal peptide    -   WBC white blood cells

What is claimed is:
 1. A composition of matter comprising: (i) amonomeric or multimeric Fc domain having a cysteine or non-canonicalamino acid substitution at one or more specifically selected conjugationsite(s) selected from D46, S48, H49, E50, E53, K55, D61, G62, Q76, Y81,K107, K121, G122, Q123, E126, R136, D137, T140, K141, N142, E169, N170,N171, K173, L179, S181, G183, D194, K195, R197, Q199, Q200, G201, N202,or S223, relative to reference sequence SEQ ID NO:599; and (ii) at leastone additional functional moiety, wherein the functional moiety isconjugated to the Fc domain through the side chain of the cysteineresidue or non-canonical amino acid residue substituted at said one ormore conjugation site(s).
 2. The composition of matter of claim 1,wherein the additional functional moiety is a pharmacologically activemoiety.
 3. The composition of matter of claim 2, wherein thepharmacologically active moiety is a polypeptide, a peptide, or apeptidomimetic moiety.
 4. The composition of matter of claim 3, whereinthe peptide is a toxin peptide.
 5. The composition of matter of claim 3,wherein the peptide comprises a cyclic peptide.
 6. The composition ofmatter of claim 2, wherein the pharmacologically active moiety is anon-peptide organic moiety.
 7. The composition of matter of claim 1,wherein the additional functional moiety is a labeled moiety comprisinga radioisotope, an enzyme, a biotinyl moiety, a fluorophore, or achromophore.
 8. The composition of matter of claim 1, wherein theadditional functional moiety is an immobilized substrate.
 9. Thecomposition of matter of claim 1, wherein the additional functionalmoiety is a half-life extending moiety.
 10. The composition of matter ofclaim 9, wherein the half-life extending moiety is a polyethyleneglycol, a copolymer of ethylene glycol, a polypropylene glycol, acopolymer of propylene glycol, a carboxymethylcellulose, a polyvinylpyrrolidone, a poly-1,3-dioxolane, a poly-1,3,6-trioxane, anethylene/maleic anhydride copolymer, a polyaminoacid, a dextran n-vinylpyrrolidone, a poly n-vinyl pyrrolidone, a propylene glycol homopolymer,a propylene oxide polymer, an ethylene oxide polymer, a polyoxyethylatedpolyol, a polyvinyl alcohol, a linear or branched glycosylated chain, apolyacetal, a long chain fatty acid, a long chain hydrophobic aliphaticgroup, an immunoglobulin Fc domain, an albumin, a transthyretin, athyroxine-binding globulin, or a ligand that has an affinity for a longhalf-life serum protein, said ligand being selected from the groupconsisting of peptide ligands and small molecule ligands; or acombination of any of these members.
 11. The composition of matter ofclaim 1, wherein the functional moiety is conjugated to the Fc domainthrough the side chain of the cysteine residue substituted at said oneor more conjugation site(s).
 12. The composition of matter of claim 1,wherein the additional functional moiety is a polymer.
 13. Thecomposition of matter of claim 1, wherein the additional functionalmoiety is polyethylene glycol.
 14. A composition of matter of theformula

wherein: F¹ is a monomer of a monomeric or multimeric Fc domain; X¹ iscovalently bound to the N-terminus of F¹ through the α-amino site of F¹;X² is covalently bound to the C-terminus of F¹ through the α-carboxysite of F¹; X³ is covalently bound to one or more specifically selectedconjugation site(s) in F¹ through the side chain of a cysteine residueor non-canonical amino acid residue added to the Fc domain bysubstitution at one or more said conjugation sites selected from D46,S48, H49, E50, E53, K55, D61, G62, Q76, Y81, K107, K121, G122, Q123,E126, R136, D137, T140, K141, N142, E169, N170, N171, K173, L179, S181,G183, D194, K195, R197, Q199, Q200, G201, N202, or S223, relative toreference sequence SEQ ID NO:599, or, if g>1, any combination of thesemembers; X¹, X², and X³ are each independently selected from-(L¹)_(c)-P⁰, -(L¹)_(c)-P¹, -(L¹)_(c)-P¹-(L²)_(d)-P²,-(L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³, and-(L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³-(L⁴)_(f)-P⁴; P⁰, P¹, P², P³ and P⁴are each independently selected from the group consisting of: i) apharmaceutically acceptable polymer or dextran; ii) a pharmacologicallyactive polypeptide, peptide, peptidomimetic, or non-peptide organicmoiety; iii) a radioisotope, an enzyme, a biotinyl moiety, afluorophore, or a chromophore; and iv) an immobilized substrate,provided that in a chain comprising more than one additional functionalmoiety, the immobilized substrate is the moiety most distal from F¹, andthere can be no more than one immobilized substrate in the chain; L¹,L², L³, and L⁴ are each independently linkers; a, b, c, d, e, and f areeach independently 0 or 1; and g is 1, 2, 3, or
 4. 15. The compositionof matter of claim 14, wherein the Fc domain comprises an IgG Fc domain.16. The composition of matter of claim 15, wherein the Fc domaincomprises an IgG1 Fc domain.
 17. The composition of matter of claim 16,wherein the IgG1 Fc domain comprises SEQ ID NO: 600 or SEQ ID NO: 603.18. The composition of matter of claim 14, wherein a=0 and b=1, or a=1and b=0, or a=1 and b=1, or a=0 and b=0.
 19. The composition of matterof claim 14, wherein X³ comprises polyethylene glycol (PEG).
 20. Thecomposition of matter of claim 14, wherein X³ has the structure-(L¹)_(c)-P¹ or -(L¹)_(c)-P¹-(L²)_(d)-P².
 21. The composition of matterof claim 14, wherein g=1 or
 2. 22. The composition of matter of claim14, wherein any of X¹, X², or X³ comprises a pharmacologically activepolypeptide, peptide, or peptidomimetic.
 23. The composition of matterof claim 22, wherein the peptide is a toxin peptide.
 24. The compositionof matter of claim 22, wherein the peptide comprises a cyclic peptide.25. A pharmaceutical composition, comprising the composition of matterof claim 1 and a pharmaceutically acceptable carrier.
 26. A compositionof matter comprising: (a) an antibody comprising a Fc domain having acysteine or non-canonical amino acid substitution at one or morespecifically selected conjugation site(s) selected from D46, S48, H49,E50, E53, K55, D61, G62, Q76, Y81, K107, K121, G122, Q123, E126, R136,D137, T140, K141, N142, E169, N170, N171, K173, L179, S181, G183, D194,K195, R197, Q199, Q200, G201, N202, or S223, relative to referencesequence SEQ ID NO:599; and (b) at least one X³ additional functionalmoiety, wherein the X³ additional functional moiety is conjugated to theFc domain of the antibody through the side chain of the cysteine residueor non-canonical amino acid residue substituted at said one or morespecifically selected conjugation site(s) in the Fc domain; and whereinX³ is selected from -(L¹)_(c)-P⁰, -(L¹)_(c)-P¹,-(L¹)_(c)-P¹-(L²)_(d)-P², -(L²)_(c)-P²-(L³)_(e)-P³, and-(L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³-(L⁴)_(f)-P⁴; P⁰, P¹, P², P³ and P⁴are each independently selected from the group consisting of: i) apharmaceutically acceptable polymer or dextran; ii) a pharmacologicallyactive polypeptide, peptide, peptidomimetic, or non-peptide organicmoiety; iii) a radioisotope, an enzyme, a biotinyl moiety, afluorophore, or a chromophore; and iv) an immobilized substrate,provided that in a chain comprising more than one additional functionalmoiety, the immobilized substrate is the moiety most distal from the Fcdomain, and there can be no more than one immobilized substrate in thechain; L¹, L², L³, and L⁴ are each independently linkers; c, d, e, and fare each independently 0 or
 1. 27. A pharmaceutical composition,comprising the composition of matter of claim 14 and a pharmaceuticallyacceptable carrier.
 28. The composition of matter of claim 26, whereinthe X³ additional functional moiety is conjugated to the Fc domain ofthe antibody through the side chain of the cysteine residue substitutedat said one or more conjugation site(s).
 29. The composition of matterof claim 26, wherein X³ comprises a pharmacologically activepolypeptide, peptide, or peptidomimetic.
 30. The composition of matterof claim 26, wherein the Fc domain is an IgG Fc domain.
 31. Thecomposition of matter of claim 30, wherein the Fc domain is an IgG1 Fcdomain.
 32. The composition of matter of claim 31, wherein the IgG1 Fcdomain comprises SEQ ID NO: 600 or SEQ ID NO:
 603. 33. A pharmaceuticalcomposition, comprising the composition of matter of claim 26 and apharmaceutically acceptable carrier.